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(Received for publication, January 19, 1996, and in revised form, May 9, 1996)
From the Clinical Pharmacology Branch, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT Treatment of SKBr3 human breast carcinoma cells
with the benzoquinoid ansamycin, geldanamycin, rapidly depletes
p185c-erbB-2 protein-tyrosine kinase.
Loss of p185c-erbB-2 is initiated by
disruption of a heteromeric complex between
p185c-erbB-2 and the 94-kDa
glucose-regulated protein, GRP94, to which geldanamycin binds avidly.
Here we report that within minutes of exposure to geldanamycin, mature
p185c-erbB-2 becomes polyubiquitinated.
Treatment of cells with the specific proteasome proteolytic inhibitor,
lactacystin, blocked geldanamycin-induced degradation of
p185c-erbB-2 and enhanced the
accumulation of polyubiquitinated
p185c-erbB-2. Following geldanamycin
and lactacystin treatment, a higher molecular weight form of
p185c-erbB-2, which likely represents
ubiquitin-p185c-erbB-2 conjugates, was
detected by anti-p185c-erbB-2
immunoblotting. Nascent p185c-erbB-2
synthesized in the presence of geldanamycin is incompletely
glycosylated and remains sequestered in the endoplasmic reticulum.
While this immature form of the protein is not ubiquitinated in the
presence of geldanamycin, its marked, drug-induced instability is
nonetheless antagonized by lactacystin. Thus, the rapid depletion of
mature p185c-erbB-2 caused by
geldanamycin and the marked, drug-stimulated decrease in half-life of
the newly synthesized protein are both mediated by the proteasome,
although only the former phenomenon involves polyubiquitination.
INTRODUCTION The c-erbB-2 proto-oncogene (also designated
HER-2/neu) encodes an 185-kDa, glycosylated transmembrane
protein-tyrosine kinase, p185c-erbB-2,
that is highly homologous to and structurally related to the cellular
epidermal growth factor receptor (1). Overexpression of
c-erbB-2 has been found in a variety of aggressive human
malignancies, and an elevated level of
p185c-erbB-2 in primary tumors is
associated with frequent relapse and an especially poor prognosis for
breast and ovarian cancers (2). Normal breast epithelial cells can be
transformed by transfection with the c-erbB-2 gene,
suggesting that p185c-erbB-2 plays a
role in the neoplastic process (3), but also hinting that inhibition of
p185c-erbB-2 function in certain human
tumors may be therapeutic.
Recently, Miller et al. (4) reported that
p185c-erbB-2 protein and its tyrosine
kinase activity are dramatically decreased by treating cells with
herbimycin (HA)1 or other benzoquinone
ansamycin antibiotics, including geldanamycin (GA). The
Streptomyces fermentation products GA and HA are inhibitors
of several protein-tyrosine kinases (5), but these ansamycins are now
thought to indirectly inhibit protein kinases in cells. In particular,
HA and GA bind strongly to heat shock protein 90 (HSP90) and disrupt
its chaperone association with pp60v-src
protein-tyrosine kinase (6) and with c-Raf-1 serine/threonine kinase
(7), resulting in reduced stability and greatly diminished levels of
these HSP90-complexed enzymes.
Depletion of p185c-erbB-2 by GA has
been reported to be mediated by a p100 protein (8), which we have
recently identified as the glucose-regulated protein, GRP94, a member
of the HSP90 family of stress proteins (9, 10). GRP94 apparently
functions to maintain the steady-state level of membrane-associated
p185c-erbB-2 by preventing its
degradation (4, 9). As a consequence of GA binding specifically to
GRP94, p185c-erbB-2 dissociates from
the heterocomplex and is subsequently rapidly degraded (9).
Having established this sequence of events, we have now investigated
the biochemistry of the proteolytic processes that are
responsible for the rapid degradation of
p185c-erbB-2 following its dissociation
from GRP94. In the present study, we show that GA rapidly stimulates
the polyubiquitination of
p185c-erbB-2 and directs its
proteolysis by the cytosolic ATP-dependent, 26 S proteasome complex. We
further demonstrate that nascent, immature
p185c-erbB-2, synthesized in the
presence of GA, is also rapidly degraded by the proteasome, but without
prior ubiquitination.
MATERIALS AND METHODS Mycoplasma-free SKBr3 human breast carcinoma
cells obtained from the American Type Culture Collection, Rockville, MD
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% heat-inactivated fetal bovine serum, 2 mM
L-glutamine, and 10 mM HEPES, pH 7.5, under
standard tissue culture conditions. Cells growing in log phase in
100-mm plates were treated with GA (1 nM to 10 µM) dissolved in Me2SO for various times (1 min to 17 h); control cells were exposed to equivalent volumes of
Me2SO alone (0.1% or less). Lactacystin (LC) was first
dissolved in water at 2 mM, then diluted in complete medium
to 10 µM, unless stated otherwise.
GA was obtained from the Developmental
Therapeutics Program, National Cancer Institute (Rockville, MD). Mouse
anti-c-ErbB-2 protein monoclonal antibodies, clone TA-1
(anti-c-neu, Ab-5, used for immunoprecipitations) and clone
3B5 (Ab-3, used for Western immunoblots) were from Oncogene Science
(Uniondale, NY). Rabbit anti-ubiquitin polyclonal antibody was from
Sigma, and mouse 4F3 anti-ubiquitin monoclonal
antibody was a gift from Dr. Linda Guarino, Texas A&M University,
College Station, TX. Other antibodies used were: rabbit anti-mouse
IgG1 (Cappel, Durham, NC) and horseradish
peroxidase-conjugated sheep anti-mouse antibody and horseradish
peroxidase-conjugated donkey anti-rabbit antibody (Amersham Life
Science). LC was purchased from Dr. E. J. Corey, Harvard University,
Cambridge, MA. All other chemicals used in this study were purchased
from Sigma.
SKBr3 tumor cells
were washed twice in ice-cold phosphate-buffered saline, then lysed on
ice with TNESV lysis buffer (50 mM Tris-HCl, pH 7.5, 1%
Nonidet P-40 detergent, 2 mM EDTA, 100 mM NaCl,
10 mM orthovanadate) with protease inhibitors: 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and
20 µg/ml aprotinin. After clearing the cell lysates of debris by
centrifuging at 14,000 × g for 20 min, protein
concentrations were determined by the bicinchoninic acid method using
bovine serum albumin as the standard (11).
p185c-erbB-2 was immunoprecipitated
from cell lysates with 2 µg of anti-c-ErbB-2 protein mAb (Ab-5)
followed by rabbit anti-mouse IgG1 (Cappel, Durham, NC)
previously bound to protein A-Sepharose beads (RAM-PAS beads). Where
indicated, SDS was added to cell lysates to a final concentration
of 1% (w/v), and following a 30-min incubation on ice and 10-fold
dilution with lysis buffer,
p185c-erbB-2 was
immunoprecipitated as usual. The proteins were eluted from their immune
complex by heating to 95 °C in reducing loading buffer (12), and
after fractionating by 8% SDS-PAGE, they were electrotransferred to
Protran nitrocellulose membranes (Schleicher & Schuell). Aliquots of
cell lysates (10-50 µg) diluted in reducing loading buffer were also
separated by SDS-PAGE and transferred to membranes for immunoblotting.
The membranes used for anti-ubiquitin Western blots were first
autoclaved in water for 20 min to insure complete denaturation of
ubiquitinated proteins.2 All membranes were
blocked with 5% fat-free dry milk in 10 mM Tris, pH 7.5, 50 mM NaCl, 2.5 mM EDTA buffer.
p185c-erbB-2 was detected by
immunoblotting with Ab-3 anti-c-ErbB-2, and ubiquitinated
p185c-erbB-2 with either anti-ubiquitin
polyclonal or monoclonal antibody, followed by the appropriate
horseradish peroxidase-linked secondary antibodies. Visualization was
by chemiluminescence (13), using a commercial kit (RenaissanceTM,
DuPont NEN). X-OmatTM AR films (Kodak) were scanned with a Foto/Eclipse
Gel Analysis system (Fotodyne), and the images were captured using
Collage 2.0 software, processed with a MacintoshTM computer (PowerPoint
3.0), and quantified using image analysis software (NIH Image
1.59).
RESULTS Within 1 h after treating SKBr3
cells with GA, an immunoprecipitable complex between
p185c-erbB-2 and GRP94 is disrupted and
p185c-erbB-2 levels rapidly decline
thereafter (8, 9). In order to determine whether
p185c-erbB-2 was ubiquitinated prior to
its degradation, cells were treated with 3 µM GA and
lysed, and p185c-erbB-2 was
immunoprecipitated using a monoclonal antibody (Ab-5) directed to an
extracellular epitope of this protein. Western immunoblots for
p185c-erbB-2 with a second anti-ErbB-2
mAb (Ab-3) verified that GA significantly decreased
p185c-erbB-2 within 1 h (Fig.
1, lanes 1-4). Separate immunoblots of
control and GA-treated samples probed with an anti-ubiquitin polyclonal
antibody revealed greater amounts of ubiquitinated
p185c-erbB-2 with increasing time of GA
exposure (Fig. 1, lanes 5-8), with little detectable
ubiquitinated p185c-erbB-2 in the
absence of GA. Ubiquitinated
p185c-erbB-2 did not appear as discrete
bands of increasing molecular mass, but instead was visualized on
immunoblots as a diffuse signal that extended upward toward the top of
the gel. This is a feature seen previously with other ubiquitinated
proteins (14) and probably occurs because a variable number of
ubiquitin molecules are added to the substrate protein during the
process of polyubiquitination (15). To verify that we specifically
detected ubiquitinated protein with the polyclonal antibody, we probed
blots from p185c-erbB-2
immunoprecipitations with a monoclonal anti-ubiquitin antibody (4F3)
(16) and found essentially the same result (Fig. 1, compare lanes
5-8 with lanes 9-12).
In the
first experiment (Fig. 1), ubiquitinated
p185c-erbB-2 protein was detected as
early as 15 min after adding GA to the cells, but the amount of
p185c-erbB-2 found on anti-ErbB-2
Western blots did not significantly decline until 60 min, suggesting
that ubiquitination of the protein occurred prior to its degradation.
To more carefully explore the relationship between polyubiquitination
and the degradation of p185c-erbB-2, we
treated cells with 3 µM GA for varying periods of time
prior to immunoprecipitating
p185c-erbB-2 from lysates. Cells were
also exposed to varying concentrations of GA for a fixed time of
2.5 h. By Western immunoblot of total lysate, it is clear that
p185c-erbB-2 levels were stable for
approximately 15-30 min after cells were exposed to GA, then declined
rapidly thereafter (Fig. 2A, lanes
1-6). In the companion Western blot from anti-c-ErbB-2
immunoprecipitations of the same samples, the ubiquitin signal was
slightly increased by GA treatment as brief as 5 min and this increase
was pronounced by 15 min (Fig. 2A, lanes 7-12).
The lowest concentration of GA that effectively decreased the level of
p185c-erbB-2 within 2.5 h was 0.5 µM (Fig. 2B, lanes 1-7), but
considerable ubiquitinated p185c-erbB-2
was detected in cells treated with 50 nM GA for 2.5 h
(Fig. 2B, lanes 8-14). 95 µg of cell lysates
were immunoprecipitated for the
p185c-erbB-2 immunoblots, and 1.0 mg of
cell lysates was precipitated for the ubiquitin immunoblots. These
results show that ubiquitination of
p185c-erbB-2 occurs rapidly after cells
are exposed to even relatively low concentrations of GA and that
ubiquitination of p185c-erbB-2 precedes
its degradation.
LC is
a potent, irreversible and specific inhibitor of at least three of the
peptidase activities of the 26 S proteasome (17). We therefore treated
cells with 10 µM LC for 1 h prior to and during
exposure to 3 µM GA for an additional 2.5 h, in
order to examine the role of the proteasome in
p185c-erbB-2 degradation. As shown in
Fig. 3, the loss of
p185c-erbB-2 was almost completely
prevented by LC pretreatment (lanes 1-4). Although
treatment of cells with LC alone had no effect on the amount of
p185c-erbB-2 (Fig. 3, lane
3), and little effect on the level of ubiquitinated
p185c-erbB-2 (Fig. 3, lane
7), co-treatment with GA and LC increased the accumulation of
ubiquitination of p185c-erbB-2 (Fig. 3,
lane 8). We also examined the effect of LC on the
ubiquitination of proteins in total cell lysates after GA and LC
treatments and found that LC alone, as expected, enhanced the detection
of ubiquitinated proteins (Fig. 3, lanes 9-12). These
results show that ubiquitin-conjugated
p185c-erbB-2 is a substrate for the
proteasome and that GA treatment accelerates the degradation of
p185c-erbB-2 by the proteasomal
pathway.
To rule out the possibility that
p185c-erbB-2 was degraded by lysosomal
proteases in addition to the ubiquitin-dependent proteasome
pathway, cells were treated with 3 µM GA alone or with 3 µM GA plus the lysosomal protease inhibitors: chloroquine
(18) or monensin (19) at 10 mM for 2 h. Neither of
these agents prevented the loss of
p185c-erbB-2 caused by GA treatment
(data not shown), indicating that GA-induced degradation of
p185c-erbB-2 was not catalyzed by
lysosomal proteases.
Miller et al. (4) have shown ansamycin
treatment does not alter mRNA levels for
p185c-erbB-2 or change the rate of
de novo synthesis of
p185c-erbB-2. We have verified these
observations and noted further that after immunoprecipitating
p185c-erbB-2 from cells treated
for 17 h with GA, the p185c-erbB-2
bands on immunoblots have a slightly lower molecular mass of ~160 kDa
(9). Together with the sensitivity of these bands to endoglycosidase H
(9), these data suggest incomplete glycosylation of the
immature protein, with its resultant sequestration in the endoplasmic
reticulum. We therefore investigated whether nascent
p185c-erbB-2 synthesized in the
presence of GA was a substrate for ubiquitination and proteasome
degradation.
Following a 17-h exposure to GA, only newly synthesized
p185c-erbB-2 (i.e.
synthesized in the presence of GA) remains, because essentially all of
the pre-existing, mature p185c-erbB-2
has been degraded (9). Preliminary experiments showed it was necessary
to immunoprecipitate p185c-erbB-2 from
as much as 1.0 mg of lysate from cells treated with GA for 17 h in
order to visualize on immunoblots the low level of the unstable,
nascent protein. For comparison, the anti-c-ErbB-2 immunoprecipitation
of as little as 50 µg of control lysate yields a strong
p185c-erbB-2 signal. Thus, it should be
noted in Fig. 4, that anti-c-ErbB-2 immunoprecipitations
from GA-treated cells were from 1.0 mg of lysate, while 95 µg of
lysate from control and LC-treated cells was precipitated. Overnight
exposure to 3 µM GA nearly eliminated
p185c-erbB-2 from cells, although a
small amount of the 160-kDa immature form of the protein could be
detected (Fig. 4, compare lanes 1 and 2). This nascent
protein is unstable and has a markedly shorter half-life than the
mature protein in untreated cells (4). The addition of LC to GA-treated
cells during the last 3 h of incubation resulted in noticeable
accumulation of the immature, 160-kDa species of
p185c-erbB-2 (Fig. 4, compare
lanes 2 and 4). As in previous experiments,
treating cells with LC alone had no effect on the level of
p185c-erbB-2 and did not alter its
apparent molecular mass (Fig. 4, compare lanes 1 and
3). However, nascent
p185c-erbB-2 was not
ubiquitinated to the extent of mature
p185c-erbB-2 following GA exposure
(Fig. 4, lane 6). In fact, ubiquitinated
p185c-erbB-2 from anti-c-ErbB-2
immunoprecipitations could be visualized only after a film exposure at
least 20 times longer than that normally used for visualizing
ubiquitinated p185c-erbB-2 after short
term (2.5 h) GA treatment. In addition, LC had only a minimal effect on
the low level of detectable ubiquitinated
p185c-erbB-2 after 17-h GA treatment
(Fig. 4, compare lanes 6 and 8). Even when an
amount of p185c-erbB-2 equivalent to
the control level was immunoprecipitated from lysate from cells treated
overnight with GA, only a trace of ubiquitinated
p185c-erbB-2 could be visualized by
immunoblotting (data not shown). In contrast, in immunoblots of
aliquots of total lysates separated by SDS-PAGE, the 17-h exposure to
GA alone or the 3-h exposure to LC alone clearly increased general
protein ubiquitination, while the combination of GA (17 h) and LC (3 h)
enhanced the polyubiquitination of proteins severalfold more than
either agent alone, showing that LC indeed stabilized ubiquitinated
proteins by preventing their degradation (Fig. 4, lanes
9-12).
In the previous
experiments, we did not observe heavier species of
p185c-erbB-2 on anti-ErbB-2 immunoblots
that would correspond to polyubiquitinated forms of
p185c-erbB-2. We therefore
immunoprecipitated p185c-erbB-2 from
500 µg of lysate (rather than the usual 50 µg) from cells treated
with 6 µM GA for 2 h and from cells treated with 6 µM GA for 2 h plus 10 µM LC for 3 h. Anti-c-ErbB-2 immunoblots of these samples revealed a higher
molecular mass form of p185c-erbB-2 in
GA-treated cells where the degradation of
p185c-erbB-2 was blocked by LC (Fig.
5A, lane 3); this upper
anti-c-ErbB-2-reacting band was completely absent in control cells
(lane 1), and only very faint in cells treated with GA alone
(lane 2). Given the approximate 200-kDa molecular mass of
the upper band, it probably represents the addition of one to two
8.6-kDa ubiquitin molecules to
p185c-erbB-2. It is possible we did not
detect other slower migrating bands representing polyubiquitinated
p185c-erbB-2, because the anti-c-ErbB-2
mAb we used may not recognize polyubiquitinated
p185c-erbB-2 due to stearic hindrance
caused by the attachment of longer multiubiquitin chains.
Polyubiquitinated p185c-erbB-2 could
easily be detected by anti-ubiquitin immunoblotting of identical
anti-c-ErbB-2 immunoprecipitations from GA-treated, as well as GA plus
LC-treated, cells (Fig. 5, compare lanes 4-6).
To rule out artifactual detection of ubiquitinated
p185c-erbB-2 resulting from the binding
of adventitious ubiquitinated proteins to
p185c-erbB-2 itself or to the RAM-PAS
beads used to immunoprecipitate
p185c-erbB-2, we added 1% SDS to
lysates from control, GA-treated, and GA plus LC-treated cells prior to
immunoprecipitating p185c-erbB-2. The
SDS treatment only slightly diminished the detection of ubiquitinated
p185c-erbB-2 immunoprecipitated from
GA-treated cells (Fig. 5B, lanes 4-6) compared
to SDS-untreated lysates (Fig. 5B, lanes 1-3),
indicating that the anti-ubiquitin immunoblots truly detected
polyubiquitinated p185c-erbB-2 rather
than nonspecific ubiquitinated proteins. When cell lysates were
sham-precipitated by omitting the primary anti-ErbB-2 mAb, only trace
amounts of ubiquitinated proteins were bound to the RAM-PAS beads, even
when lysates from cells treated with GA or the combination of GA and LC
were used (Fig. 5B, lanes 7-9).
DISCUSSION The benzoquinone ansamycins, including HA and GA, are antibiotics
isolated from Streptomyces, which rapidly destabilize and
accelerate the degradation of diverse signal transduction proteins,
including certain receptor and non-receptor tyrosine kinases (6, 8,
20, 21, 22), serine-threonine kinases (7), and mutated p53 (23). The major
cellular molecular target for these agents is the chaperone protein,
HSP90, to which GA and HA bind avidly (6). The signal-transducing
proteins are destabilized subsequent to the ansamycin-HSP90 interaction
that dissociates the chaperone multimolecular complexes, and
degradation of the normally chaperone-stabilized proteins rapidly
ensues (6, 7).
One of the receptor tyrosine kinases most sensitive to the
effects of ansamycins is
p185c-erbB-2, which is frequently
overexpressed in a high percentage of breast, ovarian, and prostate
carcinomas. Within hours after exposure to HA or GA,
p185c-erbB-2 is lost from human
tumor cells in which it is highly expressed, a phenomenon solely
mediated by drug-stimulated protein degradation (8). Although
p185c-erbB-2 does not associate with
HSP90, we have recently identified a molecular complex containing
p185c-erbB-2 and GRP94 (9), a protein
homologous to HSP90, which functions as a chaperone, within the
endoplasmic reticulum (10). A radiolabeled, photoaffinity
ansamycin derivative binds specifically to GRP94 in intact cells (but
not to p185c-erbB-2), and
destabilization of p185c-erbB-2
appears to be preceded by ansamycin-induced dissociation of a
p185c-erbB-2GRP94 heterocomplex
(9).
The results reported in the present study show that GA-induced
degradation of mature p185c-erbB-2
involves polyubiquitination of
p185c-erbB-2 and subsequent hydrolysis
by the 26 S proteasome. GA-stimulated ubiquitination of
p185c-erbB-2 occurred rapidly and was
easily detectable on anti-ubiquitin immunoblots within minutes of
adding GA to cells at 3 µM. Lower concentrations of GA
also promoted ubiquitination and loss of
p185c-erbB-2, albeit at longer exposure
times, i.e. 2.5 h with 50 nM GA (Fig.
2B) and after 18 h with 10 nM GA (data not
shown). The ubiquitination of
p185c-erbB-2 occurred prior to any
measurable decrease in p185c-erbB-2
protein levels, suggesting that conjugation of
p185c-erbB-2 to ubiquitin was a
prerequisite to its degradation.
By immunoprecipitating p185c-erbB-2
from a large quantity of lysate (500 µg, 10-fold more than usual)
from cells treated with GA or GA plus LC, we observed a higher
molecular weight form of p185c-erbB-2
(Fig. 5A). This slower migrating band of approximately 200 kDa that was prominent in the sample from GA plus LC-treated cells on
the p185c-erbB-2 immunoblot most likely
represents p185c-erbB-2 conjugated to
one or two 8.6-kDa ubiquitin moieties. We believe that higher
molecular weight forms of p185c-erbB-2,
corresponding to polyubiquitinated
p185c-erbB-2, may not be
recognized by the anti-p185c-erbB-2
immunoblotting antibody because of stearic interference by ubiquitin
homopolymeric chains. Two other anti-c-ErbB-2 antibodies gave a similar
result (data not shown). Therefore, if most of the ubiquitinated
p185c-erbB-2 in GA-treated cells is
polyubiquitinated (as one would expect for a proteasome substrate and
as the anti-ubiquitin immunoblots show), then most of the material
would be difficult if not impossible to detect by anti-ErbB-2
immunoblotting. Similar results have been obtained in the
identification of other polyubiquitinated proteins including the
cyclin-dependent kinase inhibitor, p27 (24), and the
epidermal growth factor receptor (25).
We propose that the dissociation of
GRP94-p185c-erbB-2 heterocomplexes
precedes the ubiquitination of
p185c-erbB-2, but due to technical
limitations, we cannot rule out the converse sequence of events. Not
only do the ubiquitin immunoblots detect the appearance of a signal on
what is essentially a null background (i.e. ubiquitinated
p185c-erbB-2 is only weakly detected in
untreated control cells), but the addition of many ubiquitin moieties
to p185c-erbB-2 would further amplify
the detectable ubiquitin signal. In contrast, measurement of
disappearance of GRP94-p185c-erbB-2
heterocomplexes requires detection of a signal decrease from a control
level that is maximal (i.e. untreated cells have the
greatest amount of detectable heterocomplex). For these reasons, we
believe that ubiquitination of
p185c-erbB-2 provides a more sensitive
indicator of the onset of GA action than the loss of the
GRP94-p185c-erbB-2 heterocomplex, but
given these caveats, ubiquitination of
p185c-erbB-2 may not necessarily be the
first in the sequence of events set in motion by GA.
To investigate whether the 26 S proteasome was in fact responsible for
degradation of p185c-erbB2, we treated
control and GA-treated cells with the specific and irreversible
proteasome inhibitor, LC (26). This proteasome inhibitor has been used
previously in a similar fashion to implicate involvement of the
proteasome in the processing and degradation of a mutated cystic
fibrosis transmembrane conductance regulator protein (27), and LC also
inhibits proteasome-mediated proteolytic processing of the NF- In an earlier report, we noted that prolonged GA treatment prevented
nascent p185c-erbB-2 from trafficking
to the plasma membrane, most likely because it failed to associate with
GRP94 and was not processed as usual, as indicated by an increase in
endoglycosidase H sensitivity (9). Instead, newly synthesized,
incompletely glycosylated p185c-erbB-2
remained localized predominantly within the endoplasmic
reticulum/cis-Golgi compartment (9). We therefore asked whether there
might be differential susceptibility between mature and nascent
p185c-erbB-2 to ubiquitination and
proteasome-mediated proteolysis following GA treatment. After cells
were exposed to GA for 17 h, we found nascent, newly synthesized
p185c-erbB-2 to be only minimally
ubiquitinated, even when LC was included for the last 3 h of GA
exposure to inhibit possible proteasomal degradation. Nonetheless, in
cells treated with the combination of LC and GA, the level of
incompletely glycosylated, lower molecular weight
p185c-erbB-2 was markedly enhanced
compared to GA alone. These observations imply that the immature
160-kDa p185c-erbB-2 protein remained
sequestered within the endoplasmic reticulum in the presence of GA, and
while not a substrate for ubiquitination, was still susceptible to
proteasome degradation. These observations parallel those reported in a
study of the mutated cystic fibrosis transmembrane conductance
regulator protein precursor, which fails to be converted to the mature,
fully glycosylated form, yet undergoes proteasome-mediated but
ubiquitin-independent degradation while retained within the endoplasmic
reticulum (27, 29).
Results presented in this paper and in our previous report (9) suggest
the binding of GA to GRP94, with the subsequent perturbation of mature
p185c-erbB-2-GRP94 complexes, triggers
events that target p185c-erbB-2 for
ubiquitination and proteasome-mediated degradation. Could GA merely be
accelerating a normal process of ligand induced
p185c-erbB-2 ubiquitination and
degradation? Mori et al. (30, 31) have shown that
platelet-derived growth factor In conclusion, the ability of GA to initiate biochemical events that
target p185c-erbB-2 for degradation by
the ubiquitin-dependent proteasome suggests this agent may
be useful in the biochemical dissection of the initiating mechanisms by
which certain proteins are rendered susceptible to the
ubiquitin/proteasome multicatalytic degradative pathway. Furthermore,
accumulating evidence suggests the ansamycin-targeted HSP90 and GRP94
chaperones play a hitherto unrecognized but critical role in this
process.
FOOTNOTES Acknowledgment We thank Dr. Linda Guarino for the 4F3
anti-ubiquitin antibody and Drs. Guarino and John Haley for helpful
discussions.
REFERENCES
Volume 271, Number 37,
Issue of September 13, 1996
pp. 22796-22801
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
,
Fig. 1.
GA causes rapid ubiquitination of
p185c-erbB-2.
p185c-erbB-2 protein was
immunoprecipitated from clarified TNESV lysates of control and
GA-treated (15-, 30-, and 60-min exposure to 3 µM GA)
SKBr3 cells, proteins were resolved by reducing 8% SDS-PAGE,
electrotransferred to nitrocellulose membranes, and immunoblotted for
p185c-erbB-2 and for ubiquitinated
p185c-erbB-2. For the anti-ErbB-2
immunoblot, 50 µg of lysate protein was used (lanes 1-4);
for anti-ubiquitin immunoblots (lanes 5-12), 1.0 mg of
lysate was immunoprecipitated. Lanes 1-4,
p185c-erbB-2; lanes 5-8,
ubiquitinated p185c-erbB-2 probed with
an anti-ubiquitin polyclonal antibody; lanes 9-12,
ubiquitinated p185c-erbB-2 probed with
4F3 anti-ubiquitin monoclonal antibody for verification.
Fig. 2.
Ubiquitination of
p185c-erbB-2 precedes its degradation
following GA. Cells were treated with 3 µM GA for
various times (1-60 min) or with a range of GA concentrations for a
fixed time of 2.5 h. Immunoblots for
p185c-erbB-2 and ubiquitin were
performed as described in the legend to Fig. 1. Shown in A
are anti-c-ErbB-2 (lanes 1-6) and anti-ubiquitin
(lanes 7-12) immunoblots of
p185c-erbB-2 immunoprecipitations from
lysates of cells treated with GA for the indicated times. The position
of the 200-kDa molecular mass marker is shown on the left.
In B are shown anti-c-ErbB-2 (lanes 1-7) and
anti-ubiquitin (lanes 8-14) immunoblots of
p185c-erbB-2 immunoprecipitations from
control cell lysates (lanes 1 and 8), and lysates
of cells treated with GA at 0.05 µM (lanes 2 and 9), 0.1 µM (lanes 3 and
10), 0.5 µM (lanes 4 and
11), 1.0 µM (lanes 5 and
12), 3.0 µM (lanes 6 and
13) and 10 µM (lanes 6 and
14).
Fig. 3.
The proteasomal inhibitor LC prevents
GA-induced depletion of
p185c-erbB-2. Prior to
immunoprecipitating p185c-erbB-2 from
lysates as described in the legend to Fig. 1, cells were treated with 3 µM GA alone, 10 µM LC alone, or the
combination of 10 µM LC plus 3 µM GA. LC
was added 1 h before incubating cells with GA (for an additional
2.5 h). Following SDS-PAGE and electrotransfer, membranes were
probed for p185c-erbB-2 (lanes
1-4) and for ubiquitin (lanes 5-12). For comparison,
both p185c-erbB-2 immunoprecipitations
(lanes 5-8) and 25-µg aliquots of total cell lysates
(lanes 9-12) were probed for ubiquitin. Controls are shown
in lanes 1, 5, and 9; GA-treated samples in
lanes 2, 6, and 10; LC-treated samples
in lanes 3, 7, and 11; LC- and
GA-treated samples in lanes 4, 8, and
12. The position of the 200-kDa molecular mass marker is
shown on the left.
Fig. 4.
Nascent
p185c-erbB-2 is poorly ubiquitinated
yet degraded by the proteasome. Cells were treated with 3 µM GA for 17 h in order to completely degrade mature
p185c-erbB-2 (9). LC (10 µM) was included during the last 3 h of incubation.
Following lysis, immunoprecipitation, SDS-PAGE, and electrotransfer,
nitrocellulose membranes were probed for
p185c-erbB-2 (lanes 1-4)
and for ubiquitin (lanes 5-12). Because only a small amount
of p185c-erbB-2 was found in cells
treated with GA for 17 h, differential amounts of lysates were
used to immunoprecipitate p185c-erbB-2:
100 µg of lysate from control and LC-treated cells and 1.0 mg of
lysate from cells treated with GA and the combination of GA plus LC.
The various treatments are indicated at the bottom of the
figure, where ``+'' denotes inclusion of the agent. The position of
the 200-kDa molecular mass marker is shown at the
left.
Fig. 5.
LC causes the accumulation of a higher
molecular weight form of p185c-erbB-2
after GA treatment. A, cells were treated with 6 µM GA for 2 h or GA for 2 h plus 10 µM LC for 3 h prior to
p185c-erbB-2 immunoprecipitation from
500 µg of lysates. Immunoblots for
p185c-erbB-2 (lanes 1-3)
and ubiquitin (lanes 4-6) were performed on identical
samples. Controls are shown in lanes 1 and 4,
GA-treated samples in lanes 2 and 5 and GA plus
LC-treated samples in lanes 3 and 6.
B, p185c-erbB-2 was
immunoprecipitated from untreated (lanes 1-3) and 1%
SDS-treated lysates (lanes 4-6) from control, 3 µM GA-treated (2 h), and GA plus 10 µM
LC-treated cells (2 h). For comparison, and to rule out artifactual
precipitation of ubiquitinated proteins, an anti-ubiquitin immunoblot
of samples sham-precipitated without the anti-c-ErbB-2 antibody but
with RAM-PAS beads is shown in lanes 7-9. Anti-ubiquitin
immunoblots of immunoprecipitated
p185c-erbB-2 from control cell lysates
are shown in lanes 1, 4, and 7;
GA-treated cells in lanes 2, 5, and
8; and GA plus LC-treated cells in lanes 3,
6, and 9.
B
precursor, as well as proteasomal destruction of the inhibitory I-B
regulatory protein (28). We found that LC potently blocked
GA-dependent depletion of
p185c-erbB-2, maintaining
p185c-erbB-2 at or near the control
level in the presence of GA. Furthermore, although LC itself did not
greatly affect ubiquitination of
p185c-erbB-2, the inclusion of LC
during GA treatment enhanced the accumulation of polyubiquitinated
p185c-erbB-2 that otherwise would have
been rapidly degraded. Thus, these results show the degradation of
mature, plasma membrane-localized
p185c-erbB-2 after GA treatment
proceeds predominantly, if not solely, through
ubiquitin-dependent proteasomal hydrolysis. This conclusion is
further supported by our observation that the lysosomal protease
inhibitors, chloroquine and monensin, failed to attenuate the
GA-induced loss of p185c-erbB-2. Our
findings on the fate of mature
p185c-erbB-2 following GA treatment are
quite similar to those recently reported by Sepp-Lorenzino et
al. (22) for the decline in several other tyrosine kinase
signaling receptors following HA exposure.
receptor, the epidermal growth
factor receptor, colony stimulating factor-1 receptor, and fibroblast
growth factor receptor tyrosine kinases all become polyubiquitinated as
a direct consequence of ligand binding. In addition, recently,
Galcheva-Gargova et al. (25) have verified that the
epidermal growth factor receptor is conjugated to ubiquitin following
activation by its ligand. If this hypothesis is correct, then by
analogy with p185c-erbB-2, the other
receptor signal-transducing kinases may be protected from degradation
by the formation of a ligand-sensitive stabilization complex with
GRP94, HSP90, or similar chaperone proteins. At the least, the
interaction between GRP94 and
p185c-erbB-2 might serve as a model for
an important regulatory mechanism for modulation of the protein level
of receptor tyrosine kinases. We recently reported that such
protein-chaperone interactions (in this example
pp60v-src and HSP90) can be dissociated by the
phosphorylation state of both the chaperone and the target protein,
identifying a physiologic mechanism for modulating protein-chaperone
heterocomplexes which could indirectly result from
ligand-dependent activation of the receptor (32). Such a
model of receptor regulation through phosphorylation, although
involving a chaperone complex, differs from the mechanism of
receptor-chaperone instability caused by GA, which results from a
direct interaction of GA with the chaperone rather than the
receptor.
To whom correspondence should be addressed: Clinical Pharmacology
Branch, National Cancer Institute, National Institutes of Health, Bldg.
10, Room 12N226, Bethesda, MD 20892. Tel.: 301-496-8050; Fax:
301-402-1608.
1
The abbreviations used are: HA, herbimycin; GA,
geldanamycin; GRP94, 94-kDa glucose-regulated protein; HSP90, 90-kDa
heat shock protein; LC, lactacystin; mAb, monoclonal antibody; PAGE,
polyacrylamide gel electrophoresis.
2
L. Guarino, personal communication.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT