From the
Herbimycin A is an ansamycin antibiotic isolated as an agent
that reverses morphological transformation induced by v-src.
Although herbimycin A is widely used as a tool for inhibiting multiple
tyrosine protein kinases and tyrosine kinase-activated signal
transduction, its mechanism of action is not well defined and includes
a decrease in both tyrosine kinase protein levels and activity (Uehara,
Y., Murakami, Y., Sugimoto, Y., and Mizuno, S.(1989) Cancer Res. 49, 780-785). We now show that herbimycin A induces a
profound decrease in the total cellular activity of transmembrane
tyrosine kinase receptors, such as insulin-like growth factor, insulin,
and epidermal growth factor receptors. A substantial proportion of the in vivo inhibition could be explained by an increase in the
rate of degradation. The enhanced degradation of insulin-like growth
factor-insulin receptor was prevented by inhibitors of the 20S
proteasome, whereas neither lysosomotropic agents nor general serine-
and cysteine-protease inhibitors were active in preventing receptor
degradation induced by herbimycin A. Moreover, in a
temperature-sensitive mutant cell line defective in the E1-catalyzed activation of ubiquitin, herbimycin A treatment
at the restrictive temperature did not result in the degradation of
insulin receptor. These results suggest that herbimycin A represents a
novel class of drug that targets the degradation of tyrosine kinases by
the 20S proteasome. The ubiquitin dependence of this process indicates
that this degradation of tyrosine kinases might involve the 20S
proteasome as the proteolytic core of the ubiquitin-dependent 26S
protease.
Tyrosine kinases play key roles in the regulation of a variety
of cellular processes, ranging from differentiation to malignant
transformation(1, 2, 3, 4, 5, 6, 7, 8, 9) .
The availability of specific tyrosine kinase inhibitors is a useful
tool for dissecting molecular pathways regulated by these enzymes. They
also represent a potential class of chemotherapeutic agents that
functions by inhibiting proto-oncogene products. One such inhibitor,
herbimycin A (HA),
The
establishment of autocrine and paracrine growth factor loops may play
an important role in the pathogenesis of breast cancer. Insulin-like
growth factors and transforming growth factor-
In the present study we investigated the
mechanism of HA-mediated inhibition of tyrosine kinase receptor action
in these cell lines. HA treatment effectively inhibited ligand-induced
receptor activation and tyrosine phosphorylation of downstream targets. In vitro kinase activity of the activated receptors was
inhibited by HA only slightly and required higher concentrations than
those required for the intracellular effect. We have found that in the
cell, HA induces a rapid decline in the level of tyrosine kinases
without affecting the steady-state levels of other cellular proteins.
The decrease in intracellular tyrosine kinase protein content is
secondary to induction by the drug of an increased rate of degradation.
The enhanced degradation of tyrosine kinases was found to be dependent
on the 20S proteasome and the ubiquitin-conjugating pathway. Inhibitors
of 20S proteasome proteolytic activities prevented the HA effect. Thus,
HA leads to inhibition of transmembrane tyrosine kinases by causing
their selective degradation in a process requiring the 20S proteasome,
presumably as the catalytic core of the ATP- and ubiquitin-dependent
26S protease.
As shown in Fig. 1A, insulin stimulation of
serum-starved MCF-7 cells at concentrations that stimulate IGF-IR (5
min, 1 µM) led to two major changes on phosphotyrosine
blots of total cellular protein: phosphorylation of the
The same response to HA was observed in a different breast cancer
cell line, MDA MB-468, which expresses abundant amounts of IR and
overexpresses EGFR(22, 23) . In these cells, a similar
dose- and time-dependent reduction in the steady-state levels of
tyrosine kinase receptors by HA was observed (Fig. 4). Moreover,
p56lck levels were decreased in response to HA in Colo 205
colon carcinoma cells, as was c-kit in mast cells (data not
shown). It was recently shown that the steady-state levels of erbB-2 in MDA MB-453 cells (24) and of EGFR in A431
cells (41) were also reduced in response to HA treatment. In summary,
HA does not cause a generalized effect on protein turnover, but rather
it seems to affect protein tyrosine kinases primarily.
Lysosomal
function was blocked by the lysosomotropic bases ammonium chloride and
chloroquine. In order to inhibit the proteolytic activity of the 20S
proteasome (the proteolytic core of the 26S protease), a series of
peptidyl aldehydes(19, 31)
In this report we show that HA leads to the functional
inhibition of cellular tyrosine kinase receptors by inducing their
degradation. We found that HA caused a time- and dose-dependent
reduction in the steady-state levels of IGF-IR, IR, and EGFR receptors (Fig. 3). Analysis of the steady-state levels of other cellular
proteins (including serine and threonine protein kinases) after HA
treatment revealed that the HA effect seems to be specific for tyrosine
kinases. Measurements of the kinetics of protein turnover showed that
HA enhanced the degradation of tyrosine kinase receptors (Fig. 4, A-C, and data not shown) with no significant changes in the
rate of protein synthesis (Fig. 4D). Analysis of the
molecular pathways involved in this effect suggested that the 20S
proteasome and the ubiquitin pathway of protein degradation were
involved ( Fig. 5and Fig. 6). We employed a set of
inhibitors of a variety of cellular proteases and of lysosomal function
to study this phenomenon (Fig. 5). We found that neither
inhibition with the nonspecific serine- and cysteine-protease
inhibitors PMSF, leupeptin, and E64d, nor inhibition of the
Ca
We also employed a set of peptidyl aldehyde inhibitors designed to
inhibit the catalytic activities of the 20S proteasome ()(19) . The 20S proteasome or multicatalytic
proteinase complex is a large cytosolic and nuclear proteinase which is
thought to constitute the proteolytic core of the ubiquitin-dependent
proteolytic machinery(27, 28, 31, 33) .
It is distinguished by the wide specificity of the proteolytic
reactions it catalyzes. At least five distinct proteolytic components
of the 20S proteasome have been described(31) . These are
referred to as tryspin-like, chymotrypsin-like, peptidyl-glutamyl
peptide hydrolyzing, small neutral amino acid-preferring, and BrAAP.
Each of these activities is catalyzed by a physically distinct subunit
or a set of subunits of 20S proteasome, not by a single site with a
broad range of substrate specificities(31, 33) . A
series of peptidyl aldehyde inhibitors for the 20S proteasome were
designed based on sequences derived from known 20S proteasome
substrates of the BrAAP component. These inhibitors possess a
C-terminal aldehyde, which by reacting with a nucleophilic center in
the active site of a protease forms a hemiacetal or thiohemiacetal,
which is thought to mimic the putative transition state of peptide bond
hydrolysis(19) . These peptidyl aldehyde inhibitors effectively
blocked protein degradation catalyzed by the 20S proteasome in
vitro and led to the accumulation of ubiquitin-protein conjugates
in cells (19). Conversely, the peptidyl alcohol precursors of these
inhibitors had no effect in either setting(19) , thus confirming
the necessity of the aldehyde group for inhibition. We observed that
there was a dose-dependent accumulation of ubiquitin-protein conjugates
in cells treated with the aldehyde inhibitors, with no change in
response to the inactive alcohols (Ref. 19 and data not shown). When a
combination of HA and each of these compounds were added to cells, we
observed that the enhanced degradation of tyrosine kinases was
prevented (Fig. 5). Moreover, when a series of peptidyl aldehyde
inhibitors were employed, we observed a direct relationship between the
efficacy of these compounds in preventing the HA effect and their
potencies as inhibitors of the BrAAP activity of the 20S proteasome (Fig. 5, Ref. 19, and data not shown). It is important to note
that, in the absence of HA, the BrAAP inhibitors had no effect per
se on the levels of tyrosine kinases. Also, none of the
lysosomotropic agents or general serine-and cysteine-protease
inhibitors tested affected protein degradation by the 20S proteasome at
the range of concentrations employed. Therefore, our data point to a
model in which HA leads to the degradation of tyrosine kinases in a
process that involves their selective targeting to the 20S proteasome.
The best studied function of the 20S proteasome is that of the
proteolytic core of the ATP-dependent 26S protease which primarily
catalyzes ubiquitin-dependent degradation of
proteins(27, 28, 31) . Therefore, we studied
whether ubiquitination was required for the HA effect.
Immunoprecipitated IGF-IR from HA-treated cells is associated with
ubiquitin as assessed by Western blotting with an anti-ubiquitin
antibody (data not shown). However, it was difficult to produce a blot
of high quality, due to the poor reactivity of the anti-ubiquitin
antibodies employed and the rapid loss in IGF-IR in HA-treated cells.
Therefore, we used a conditional mutant cell line to test whether
ubiquitination played a role in the HA-induced degradation of these
receptors. As shown in Fig. 6, in a temperature-sensitive cell
line (35) deficient in the ubiquitin-activating enzyme E1 (the first enzyme in the ubiquitin conjugation
pathway)(27, 29) , HA was unable to induce the
degradation of transfected IR under restrictive conditions, while
effectively reducing IR levels at the permissive temperature. Although
this result does not unequivocally prove that ubiquitination of the
tyrosine kinase occurs, it indicates that a functional ubiquitin
pathway is necessary for HA to induce degradation. We are currently
studying the molecular mechanism of this effect in a cell-free system.
A proposed mechanism for the in vitro inhibition of
tyrosine kinase activity by HA involves the formation of an adduct
between a reactive cysteine residue in the kinase with the benzoquinone
group of the drug, thereby leading to the inactivation of the
enzyme(3, 6) . This model was based on the observations
that sulfhydryl compounds such as DTT, 2-mercaptoethanol, glutathione
(reduced form), or cysteine inactivated HA, whereas methionine,
cystine, or oxidized glutathione had no effect (3, 6, and Fig. 2). Comparative sequence analysis of protein kinases
revealed the presence of 2 conserved Cys in the kinase domain of
receptor and cytosolic tyrosine kinases, which are conspicuously absent
from most serine/threonine protein kinases(36, 37) . The
primary role of these Cys residues in catalysis was illustrated in
mutants generated for p56
A model for the cellular effect of HA
could be proposed wherein the ubiquitin conjugating system might
recognize the HA
Standard one or
three letters abbreviations for amino acids are used.
We thank Dr. L-H. Wang for providing us with
anti-IGF-IR antibody, Drs. J. Mendelsohn and K. Masui for the anti-EGFR
monoclonal antibody 225, and Dr. R. Kulka for the ts20 cell line. We
gratefully acknowledge Drs. M. Orlowski and C. Cardozo for support and
Dr. J. Rothman for a critical review of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)is a benzoquinonoid
ansamycin antibiotic originally isolated as an agent capable of
reverting the transformed phenotype of Rous sarcoma virus-infected
normal rat kidney cells(1) . Cellular and biochemical analysis
of HA-treated cultures indicated that the morphological reversion was
accompanied by a decrease in the kinase activity of the oncoprotein,
together with increased turnover of
pp60
(4, 10) . HA also effectively
reversed the morphologic transformation induced by other tyrosine
kinase oncogenes such as yes, fps, abl, erbB, and ros but not by ras, raf, and myc(2) .
Moreover, HA inhibited the tyrosine kinase activities of several src-family members and of p210
in
vitro(3, 5, 6, 11) , whereas no
effect was observed on the in vitro or intracellular catalytic
activities of serine/threonine kinases such as protein kinase A,
protein kinase C, or
Raf-1(3, 5, 6, 11) .
are potent mitogens
for breast cancer cells, and tyrosine kinase receptors such as IGF-IR,
EGFR, and erbB-2 are overexpressed in a significant percentage
of tumors(12, 13) . Moreover, several breast cancer cell
lines are dependent on the activity of growth factor receptors for the
maintenance of their transformed
state(14, 15, 16) . Therefore, these tyrosine
kinase receptors are potential targets for inhibitors such as HA. We
have found that the breast cancer cell lines MCF-7 and MDA MB-468
(which express high levels of IGF-IR and EGFR, respectively) are
profoundly inhibited by HA with respect to anchorage-dependent and
-independent growth.
Materials
Herbimycin A (HA) was purchased from
Life Technologies, Inc., dissolved in MeSO to 1 mg/ml,
aliquoted, and kept frozen at -20 °C.
[
-
P]ATP (3000 Ci/mmol) and
S-Protein labeling mix (L]
S]methionine, 1200 Ci/mmol) were
purchased from Dupont NEN. Electrophoresis and electrotransfer reagents
were from Fisher, Sigma, and Life Technologies, Inc. All other reagents
were analytical grade and obtained from standard suppliers.
Antibodies
Monoclonal antibodies, anti-human
IGF-IR antibody (IR-3), and anti-human IR (Ab-1) were from
Oncogene Science, anti-phosphotyrosine antibody PT-66 was from Sigma,
and anti-phospholipase C
was from UBI. Polyclonal antibodies
against EGFR, GAP, p85-PI3 kinase, and SHC were purchased from UBI, and
anti-ubiquitin antibody was from Sigma. A polyclonal antibody that
recognizes the
subunit of IGF-IR in immunoblots was kindly
provided by Dr. L.-H. Wang (Mt. Sinai Medical Center, New York),
whereas a monoclonal antibody against human EGFR (mAb 225) was from
Drs. J. Mendelsohn and H. Masui (Memorial Sloan-Kettering Cancer
Center). A polyclonal antisera for baculovirus-produced human IR
-subunit was raised by one of us (D. E. L., Ref. 17).
Protease Inhibitors
The peptidyl aldehyde
inhibitors for the 20S proteasome were synthesized by the oxidation of
corresponding alcohols by the procedure of Pfitzner and Moffat (18) as modified by Vinitsky et al.(19) . Their
synthesis and characterization of their effect in vitro and in vivo is described in Ref. 19. The following 20S proteasome
inhibitors were employed in this study ():
Bz-Gly-Pro-Ala-phenylalaninal (GPAF-al), Bz-Pro-Gly-Ala-leucinal
(PGAL-al), Bz-Gly-Pro-Phe-valinal (GPFV-al), Bz-Gly-Pro-Ala-leucinal
(GPAL-al or 2al), Bz-Gly-Pro-Phe-leucinal (GPFL-al or 1al),
Bz-Gly-Ala-Phe-leucinal (GAFL-al or 4al), and Bz-Gly-Leu-Ala-leucinal
(GLAL-al or 5al). All inhibitors were dissolved in MeSO at
2 mM and stored at -20 °C. In order to extend the
half-life of these inhibitors in vivo, cultures were
simultaneously treated with Bz-Pro-prolinal (PP-al), a peptidyl
aldehyde inhibitor of prolyl endopeptidase(20) .
Phenylmethylsulfonyl fluoride (PMSF), leupeptin, E64d, and calpain
inhibitor II were from Sigma and Calbiochem.
Cell Culture
The human breast cancer cell lines
MCF-7 and MDA MB-468 cells were from the American Type Culture
Collection, Rockville, MD. Cultures were maintained in DME/F12 (1:1)
supplemented with 5% heat-inactivated fetal bovine serum (Gemini
Bioproducts), 2 mM glutamine, and 50 units/ml each of
penicillin and streptomycin, in a humidified 5% CO/air
atmosphere at 37 °C. The temperature-sensitive E1-ts20
mutant cell line was provided by Dr. R. Kulka (The Hebrew University of
Jerusalem). Cultures were maintained at 32 °C in RPMI 1640
supplemented with 10% fetal bovine serum, glutamine, and antibiotics.
Transfections
ts20 cells were co-transfected with
a vector for human insulin receptor under the control of the
cytomegalovirus promoter (pCMV-hIR(21) ) and a vector that
confers geneticin resistance (pSV-neo). Transfection was performed with
Lipofectin (Life Technologies, Inc.) according to the
manufacturers' instructions. Pooled populations of stable
transfectants were selected after 2 weeks of exposure to geneticin (0.5
mg/ml) and maintained under selective pressure.
Immunoprecipitation and Immunoblotting
Following
experimental treatments, monolayers were washed twice with
phosphate-buffered saline, and then either dissolved directly into
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer or
solubilized in lysis buffer (50 mM Tris-HCl, pH 7.5, 1%
Nonidet P-40, 150 mM NaCl, 1 mM NaV0
, 40 mM NaF, and 10 µg/ml
each of leupeptin, aprotinin, and soybean trypsin inhibitor) (750
µl/100 mm-dish)(22) . Cell lysates were centrifuged at
15,000
g for 10 min at 4 °C in a microfuge
(Eppendorf), and the detergent solubilized proteins were
immuno-precipitated with appropriate antibodies. Immunocomplexes were
collected on protein A-Sepharose beads (Pharmacia), washed three times
with lysis buffer, and subjected to SDS-PAGE. Gels were transferred
onto nitrocellulose membranes and subjected to immunoblotting and
detection via chemoluminescence (ECL, Amersham Corp.).
In Vitro Kinase Assays
IGF-IR immunoprecipitates
from insulin-treated cells were washed twice in lysis buffer followed
by a wash in lysis buffer lacking phosphatase inhibitors (NaF and
NaVO
). Washed immunoprecipitates were then
incubated for 1 h at room temperature with either herbimycin A or
Me
SO in 40 µl of kinase buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl
, 10 mM MnCl
, 100 mM NaCl, 0.1% Triton X-100) in the
presence or absence of 1 mM DTT. In vitro kinase
reactions were initiated by the addition of 10 µCi of
[
-
P]ATP/reaction plus unlabeled
ATP to 90 µM. Reactions were carried out for various times
(1, 5, 10, 15, and 30 min) at room temperature and stopped by the
addition of 10 µl of 5
SDS-PAGE sample buffer.
Phosphorylated products were separated by SDS-PAGE; gels were
Coomassie-stained, dried, and autoradiographed. Band quantitation was
performed using a FUJIX PhosphorImager with MacBAS-1000 software.
Pulse and Pulse-chase Experiments
In order to
study the effect of HA on protein synthesis, MCF-7 cells were
pulse-labeled with [S]methionine (100
µCi/ml, 1, 200 Ci/mmol) in methionine-free media for increasing
time periods in the presence or absence of HA or its carrier
(Me
SO). Conversely, to assess the effect of HA on protein
degradation, MCF-7 cells were prelabeled to isotopic equilibrium with
[
S]methionine (100 µCi/ml, 1, 200 Ci/mmol)
in methionine-free media for 24 h and chased with unlabeled methionine
(150 µg/ml) for a subsequent 24-h period. One h into the chase,
cultures received either herbimycin A (5 µg/ml) or Me
SO
to 0.5%, and these conditions were maintained until the completion of
the chase. Lysates were made at different time points in both types of
experiments, and samples containing equal protein content were
immunoprecipitated for IGF-IR as described above. Gels containing
radiolabeled samples were Coomassie Blue-stained, fluorographed by
impregnation with EN
HANCE (DuPont NEN), dried, and exposed
to x-ray film. Band quantitation was performed using a FUJIX
PhosphorImager with MacBAS-1000 software.
Signaling via Receptor Tyrosine Kinases Is Antagonized
in Vivo by Herbimycin A
IGFs and high concentrations of insulin
activate IGF-IR and are mitogenic for MCF-7 cells under serum-free
conditions (22). Treatment of MCF-7 cells with HA not only blocked
insulin- and IGF-I-induced mitogenesis induced by activation of IGF-IR,
but also inhibited proliferation induced by 10% fetal bovine serum
(data not shown). This inhibitory effect was dose dependent and
observed with HA concentrations as low as 10 ng/ml (17.4 nM)
(data not shown). In order to understand the antiproliferative effect
of HA in these cells, we first examined whether it impaired IGF-IR
function.
-subunit
of the receptor (
100 kDa) and of a family of insulin receptor
substrates (IRS) with
185 kDa (Fig. 1). Preincubation of the
cells with increasing concentrations of HA caused a dose-dependent
decrease in the phosphotyrosine content of those insulin-responsive
substrates (Fig. 1A). Immunoprecipitation of the IGF-IR
followed by immunoblotting with an anti-phosphotyrosine antibody
confirmed the identity of the 100 kDa band as the
-subunit of the
receptor (Fig. 1B). We have previously observed that in
MCF-7 cells, the p120-rasGTPase-activating protein (GAP) becomes
tyrosine phosphorylated following insulin treatment.
(
)Tyrosine phosphorylation of GAP by insulin was also
reduced in a dose-dependent manner following treatment with herbimycin
A (Fig. 1C). Therefore, HA led to a dose-dependent
inhibition of insulin-activated IGF-IR kinase activity and biological
response as measured by phosphorylation of substrates and mitogenesis.
Figure 1:
Herbimycin
A inhibits tyrosine phosphorylation in response to insulin in MCF-7
cells in a dose-dependent manner. MCF-7 cells were serum-deprived for
24 h in the presence of increasing concentrations of herbimycin A in
MeSO (0.1, 0.5, 1, 5, and 10 µg/ml). The final
Me
SO concentration in all cultures was 0.5%. Cells were
subsequently stimulated or not (Control) with 1 µM insulin
for 5 min and detergent-solubilized proteins were immunoprecipitated
for IGF-IR (B) or for GAP (C). Immunoprecipitates and
total lysate protein (A) were analyzed via SDS-PAGE,
transferred onto nitrocellulose, and immunoblotted for phosphotyrosine
content followed by detection via chemioluminescence. The positions of
the IGF-IR receptor
-subunit, pp185-IRS-1, GAP, and the
GAP-associated proteins p190 and p62 are indicated with arrows on the right whereas the mobility of molecular weight markers are
indicated on the left by their corresponding M
.
Herbimycin A Inhibits IGF-IR Kinase Activity in
Vitro
We investigated whether the ligand-activated tyrosine
kinase activity of the IGF-IR was affected by the drug in in vitro kinase assays. Immobilized IGF-IR, immunoprecipitated from
insulin-stimulated MCF-7 cells, was preincubated with 5 µg/ml of HA
for 60 min prior to kinase assays. In the absence of DTT, 5 µg/ml
(8.7 µM) of HA decreased the rate of IGF-IR
autophosphorylation to approximately 50% of that obtained for vehicle
alone (MeSO) (Fig. 2, A and BversusC and D). No inhibition was
observed in the presence of reducing agents (Fig. 2, C and D), which supports the previous finding that
sulfhydryl groups inactivate HA(3) . Although we were able to
observe a direct effect of HA on the IGF-IR kinase in vitro, the concentrations required to observe this effect were at least
100-fold higher than those required to inhibit receptor function in the
intact cell (Fig. 1).
Figure 2:
IGF-IR kinase activity is inhibited in
vitro by herbimycin A in the absence of reducing agents. IGF-IR
was immunoprecipitated from lysates of insulin-stimulated MCF-7 cells
with IR3, a monoclonal antibody for the
-subunit of the human
receptor. Immunocomplexes bound to protein A-sepharose beads were
preincubated for 1 h at room temperature in the presence of either
Me
SO or 5 µg/ml herbimycin A in a standard in vitro kinase buffer supplemented or not with 1 mM DTT. The
final Me
SO concentration in both conditions was 2.5%. In vitro kinase reactions were carried out at room temperature
for different times, terminated by addition of SDS-PAGE sample buffer
and phosphorylated proteins were analyzed by SDS-PAGE followed by
autoradiography. IGF-IR autophosphorylation was quantitated with a
FUJIX PhosphorImager, by measuring the extent of
P
incorporation into the
-subunit of the receptor in the absence and
presence of DTT.
Herbimycin A Induces a Dose- and Time-dependent Decrease
in the Steady-state Levels of Receptor Tyrosine Kinases in Breast
Cancer Cells
The difference in concentrations required for
inhibition of receptor tyrosine kinases in vitro and in the
cell suggests either that intracellular HA is converted into a more
potent inhibitor, or that other mechanisms of inhibition predominate.
As shown in Fig. 3, HA led to a dose- and time-dependent
reduction of the cellular content of IGF-IR (precursor as well as
mature receptor) in MCF-7 cells, as detected on immunoblots of total
cellular protein or of immunoprecipitated receptor. This effect was
pronounced at 0.1 µg/ml of HA (Fig. 3A), and
relatively rapid, noticeable after 4 h, and complete by 24 h (Fig. 3B).
Figure 3:
Herbimycin A treatment leads to a decrease
in the steady-state levels of tyrosine kinases in a time- and
dose-dependent fashion, without affecting the levels of other
(non-tyrosine kinase) cellular proteins. MCF-7 or MDA MB-468 cells were
treated for 24 h with increasing concentrations of HA (A and D) as described for Fig. 1, or for different time periods with
5 µg/ml of the drug (B and E). Total cellular
lysates and immunoprecipitates for IGF-IR (A and B)
or immunoprecipitates for IR and EGFR (D) were immunoblotted
with a specific antibodies for each receptor. Lysates of MCF-7 cells
treated for 24 h with increasing HA concentrations were immunoblotted
for phospholipase C-, GAP, the p85 subunit of PI3K, and SHC with
specific antisera, as described for Fig.
1C.
To test whether this reduction in the
receptor levels was due to a generalized effect on protein metabolism,
we analyzed the effect of HA on the steady-state levels of other
cellular proteins. The profile of
[S]methionine-labeled total proteins revealed
that there was no overall decrease in protein levels induced by HA
treatment (not shown). Western blots for p85-phosphatidylinositol
3`-kinase (PI3K), phospholipase C-
, SHC, and GAP (Fig. 3C) demonstrated that the cellular abundance of
this group of proteins was not affected by the presence of HA in MCF-7
cells. The cellular abundance of other proteins such as
- and
-catenin, E-cadherin, and of serine/threonine protein kinases such
as cdk2, erk-1, and erk-2 was also not affected by HA (not shown).
Figure 4:
Herbimycin A induced an increase in the
rate of receptor tyrosine kinase degradation. In order to assess the
effect of HA on protein degradation, MCF-7 cells were prelabeled to
isotopic equilibrium with [S]methionine (100
µCi/ml, 1, 200 Ci/mmol) in methionine-free media for 24 h and
chased with unlabeled methionine (150 µg/ml) for a subsequent 24-h
period (t = 0 h). One h into the chase (t = 1 h), cultures received either herbimycin A (5 µg/ml)
or Me
SO to 0.5%, and these conditions were maintained until
the completion of the chase. Lysates made at different time points (t = 2, 3, 6, 12, and 24 h) were immunoprecipitated for
IGF-IR. The IGF-IR precursor, as well as the mature
- and
-subunits, were identified by SDS-PAGE followed by autoradiography (A) and quantitation of the levels of
[
S]methionine incorporated into each species
were measured with a FUJIX PhosphorImager (B and C).
To analyze the effect of HA on protein synthesis (D), MCF-7
cells were pulse-labeled with [
S]methionine (100
µCi/ml, 1, 200 Ci/mmol) in methionine-free media for increasing
time periods, and the specific incorporation into the
-
precursor (arrow) for the IGF-IR was quantitated as described
above.
Herbimycin A Treatment Induced an Accelerated Rate of
Receptor Tyrosine Kinase Degradation
Changes in steady-state
levels can be accomplished by altered rates of synthesis or
degradation. Equilibrium labeling of MCF-7 cells with
[S]methionine followed by chase with cold
methionine in the presence or absence of HA was used to measure the
effect of the drug on IGF-IR degradation. As shown in Fig. 4, A-C, HA induced an accelerated rate of IGF-IR degradation in
MCF-7 cells. The apparent half-life of both receptor subunits (the
extracellular, ligand-binding
-subunit and the intracellular,
tyrosine kinase-containing
-subunit) was shortened from more than
24 h in control cultures to approximately 6-7 h in the presence
of the drug. Similar results were observed for the EGFR in MDA MB-468
cells (data not shown). On the other hand, no significant differences
in the rate of [
S]methionine incorporation into
IGF-IR were observed in pulse-labeling experiments, whether or not HA
was present (Fig. 4D). Thus, tyrosine kinase synthesis
is not appreciably affected by HA. Therefore, the rapid decrease in
receptor tyrosine kinase steady-state levels observed upon HA treatment
is due to an increased rate of receptor degradation.
Herbimycin A-induced Receptor Tyrosine Kinase Degradation
Involves the 20S Proteasome
Most intracellular protein
degradation is catalyzed by lysosomal proteases (25, 26) or by the ubiquitin- and ATP-dependent 26S
protease complex(27, 28, 29, 30) . We
employed a series of inhibitors of the lysosomal and
ubiquitin-dependent proteolytic pathways to determine the mechanism by
which HA induces receptor degradation. These experiments were performed
in the absence of receptor ligand in order to exclude ligand-induced,
lysosome-dependent degradation(25, 26) , a process
involved in the down-regulation of activated receptors.
(
)was
used (). The sequences of these inhibitors are based on
known substrates of the 20S proteasome; the presence of a C-terminal
aldehyde is required for inhibition of peptide bond
hydrolysis(19) . Peptidyl aldehyde inhibitors were shown to
block protein degradation catalyzed by the 20S proteasome in vitro and the 26S protease-mediated intracellular degradation of
ubiquitinated proteins, whereas the peptidyl alcohols from which they
were derived did not(19) .
The most effective
inhibitors of the 20S proteasome are GAFL-al (4al) and GLAL-al (5al)
which strongly inhibit all of its five proteolytic components.
When these inhibitors were added to cells together with HA they
completely prevented the enhanced degradation of IGF-IR (Fig. 5, A and B). We next employed a series of peptidyl
aldehyde inhibitors of the branched-chain amino acid preferring (BrAAP)
component of the 20S proteasome, which is thought to be the major
factor in protein degradation by this protease(19) . As shown in Fig. 5B, the potencies of the peptidyl aldehyde
inhibitors of the BrAAP component in reverting the HA-induced IGF-IR
degradation are directly correlated with their ability to inhibit that
component in vitro(19) .
That correlation
also held for the ability of the inhibitors of BrAAP to affect
accumulation of ubiquitin-protein conjugates in treated cells (not
shown and 19). The most potent inhibitor from the series, GPFL-al, was
able to revert IGF-IR levels to 75% of the levels from untreated
controls (Fig. 5B). Bz-Pro-prolinal (PP-al), a peptidyl
aldehyde inhibitor of prolyl endopeptidase (20) used to prolong
the half-life of the peptidyl aldehyde inhibitors, had no effect on the
levels of IGF-IR. Lysosomotropic agents or the inactive peptidyl
alcohol analogs were also without effect in preventing the HA-induced
IGF-IR degradation (Fig. 5, A and B). Moreover,
the nonspecific serine- and cysteine-protease inhibitors PMSF,
leupeptin, and E64d, and calpain inhibitor II (a potent inhibitor of
cytosolic calpains and of lysosomal cathepsin
B(25, 26, 30) ) were also ineffective in
restoring IGF-IR levels in HA-treated cultures (Fig. 5, A and B). None of these general protease inhibitors affects
the rate of protein degradation by the 20S proteasome at the range of
concentrations employed here. Identical results were obtained for IR in
MDA MD-468 cells (data not shown). These results imply that HA-induced
degradation of IGF-IR and IR is catalyzed by the 20S proteasome.
Figure 5:
IGF-IR
degradation induced by herbimycin A is dependent on the 20S proteasome. A, MCF-7 cells were treated with 1 µg/ml HA for 9 h in the
presence or absence of lysosomotropic agents, general serine- and
cysteine-protease inhibitors (left panel) or with a series of
peptidyl aldehyde inhibitors for the 20S proteasome (right
panel). Lanes 1 and 7, untreated controls; lanes 2 and 8, HA alone; all others HA plus:
NHCl (10 mM, lane 3), chloroquine (0.5
mM, lane 4), leupeptin (175 µg/ml, lane
5), PMSF (2 mM, lane 6), 20S proteasome
inhibitors 4-al (GAFL-al), 5-al (GLAL-al), 1-al (GPFL-al) and 2-al
(GPAL-al) (50 µM, lanes 9-13). Lanes 14 and 15 are samples from HA-treated cultures
simultaneously treated with the inactive peptidyl-alcohols 5-ol
(GLAL-ol) and 1-ol (GPFL-ol) (50 µM, lanes 14 and 15). All cultures were treated with
benzyloxycarbonyl-prolyl-prolinal (PP-al), a prolyl endopeptidase
inhibitor, included to prolong the half-life of the 20S proteasome
inhibitors in vivo. PP-al by itself had no effect on the
steady-state levels of tyrosine kinases. B, densitometric
analysis of IGF-IR receptor levels from at least three independent
experiments were combined in a graph and expressed as percentage of
IGF-IR recovered ± S.E. The K values for the BrAPP
component for five of these 20S proteasome inhibitors were from
Vinitsky et al. (19). Inhibitors which were employed but not
shown in A are GPFV-al, PGAL-al, GPAF-al, E64d, and calpain
inhibitor II, all at 50 µM. Treatment of MCF-7 cells with
any of these compounds in the absence of HA did not affect the basal
steady-state levels of IGF-IR.
Degradation of Tyrosine Kinases Induced by Herbimycin A
Is Dependent on the Ubiquitin-conjugating System
The 20S
proteasome is proposed to be involved in ubiquitin-dependent and
-independent
proteolysis(27, 28, 33, 34) . In order
to discriminate between these two possibilities, we decided to
investigate whether ubiquitination of tyrosine kinases was necessary
for their sensitivity to HA. Experiments to detect ubiquitinated
receptors in immunoprecipitates failed to provide clear results (see
``Discussion''). We used a temperature-sensitive mutant cell
line defective in E1-catalyzed ubiquitin activation (35) to determine the ubiquitin requirement of the HA effect.
Activation of ubiquitin by E1 is an essential step for all
ubiquitin-dependent processes(27) , and at the restrictive
temperature, cells defective in this step are unable to carry on
protein ubiquitination(35) . ts20 cells were stably transfected
with human IR (pCMV-hIR) (21) and selected for geneticin
resistance. Pooled populations were treated with or without HA for 5 h
at either 25 or 37 °C. At the permissive temperature, HA induced
the degradation of IR; however at 37 °C, the degradation of IR was
greatly prevented (Fig. 6). Densitometric analysis indicated that
at 25 °C IR levels were reduced by 77% in the presence of HA,
whereas at 37 °C IR levels were decreased by only 13%. The
steady-state levels of total cellular ubiquitin-protein conjugates were
markedly decreased at 37 °C, indicative of an impaired E1
function (Fig. 6). The dependence of HA-induced degradation on
the ubiquitin-conjugating system together with its sensitivity toward
20S proteasome inhibitors suggest that exposure of protein tyrosine
kinases to HA leads to their selective degradation by the ubiquitin-
and ATP-dependent 26S protease.
Figure 6:
Receptor tyrosine kinase degradation
induced by herbimycin A is dependent on the presence of an active
ubiquitin pathway. Temperature-sensitive E1 ts20 mutants
transfected with the human IR were pretreated for 4 h at either 25 or
37 °C and subsequently treated with HA (1 µg/ml) or
MeSO (0.01%) in serum-free media for 5 h. The levels or IR
were measured by immunoprecipitation and immunoblotting (left
panel), and the levels of total ubiquitin-protein conjugates were
analyzed by Western blots of lysate protein with anti-ubiquitin
antibody (1:500, Sigma) (right
panel).
-dependent protease calpain with calpain inhibitor
II (which is also a potent inhibitor of the lysosomal protease
cathepsin B) (25, 26, 30) were effective in
preventing the HA-induced degradation of tyrosine kinases. Moreover,
blockade of lysosomal function with ammonium chloride or chloroquine at
concentrations capable of preventing ligand-activated EGFR
down-regulation via the endocytic pathway were also incapable of
preventing the HA effect. Therefore, we conclude that the observed
degradation of tyrosine kinases induced by HA does not occur via the
lysosomal pathway or by cytosolic proteases such as calpains. It is
important to note that although activated receptors are degraded by the
lysosome(26) , the experiments presented here were conducted in
the absence of activating ligands in order to prevent activation of
this pathway, which would complicate the analysis of the HA effect.
(38) . Cys to
Ala p56
mutants in the catalytic domain, at
positions 464 and 475, abolished catalytic activity in vitro as well as in the cell. Furthermore, C475A was highly unstable,
exhibiting a half-life six times shorter than the wild type
protein(38) . Whether this accelerated turnover of the mutant
proteins was due to a profound change in conformation or whether it
involved a mechanism similar to that induced by HA (see below) is
currently under investigation.
protein complex itself or a motif in the protein
exposed by HA binding. In this regard, it has been recently
demonstrated that geldanamycin, a HA analog, inhibited the formation of
the hsp90
v-src complex(39) . Whether this event
is sufficient to target tyrosine kinases for conjugation with ubiquitin
and subsequent proteolysis is unknown. Nevertheless, the inducibility
of tyrosine kinase degradation by the 20S proteasome and its dependence
on the ubiquitination pathway will allow the study of the elements of
the proteolytic system involved in the cells response to HA. The
results presented here suggest that induction of receptor tyrosine
kinase degradation is an important component of the mechanism by which
HA inhibits the activity of transmembrane and cytosolic tyrosine
kinases in the cell. During the preparation of this article, Murakami et al.(32) reported the enhanced degradation of EGFR
induced by HA in A431 cells. They showed that degradation occurred by a
lysosomal-independent pathway, as lysosomotropic agents had no effect
of preventing EGFR loss. In this report, we show that degradation is
dependent upon the 20S proteasome and on the ubiquitin-conjugating
system. Induction of degradation of a specific class of proteins
represents a novel mechanism for drug action and identifies an agent,
HA, which may represent a prototype of this new class of drugs. It also
affords a strategy for causing cytotoxicity in tumors whose transformed
phenotype is dependent on the presence of activated tyrosine kinases.
Table: 20S proteasome inhibitors
SO, dimethyl
sulfoxide.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.