(Received for publication, July 18, 1995; and in revised form, September 12, 1995)
From the
The platelet-derived growth factor -receptor undergoes
polyubiquitination as a consequence of ligand binding. We have
previously reported that ligand-induced ubiquitination of the receptor
plays a negative regulatory role in its mitogenic signaling possibly by
promoting the efficient degradation of the ligand-activated receptor
(Mori, S., Heldin, C.-H., and Claesson-Welsh, L.(1993) J. Biol.
Chem. 268, 577-583). In the present study, we have examined
effects of different kinds of cell-penetrating proteasome inhibitors,
including substrate-related peptidyl aldehydes,
Cbz-Ile-Glu(O-t-Bu)-Ala-leucinal (where Bu is butyl
and Cbz is benzyloxycarbonyl) (PSI) and Cbz-Leu-Leu-norvalinal (MG115),
and a Streptomyces metabolite lactacystin, on degradation of
the receptor in intact cells with the aim of evaluating the role of the
receptor ubiquitination in the proteasome-dependent proteolytic
process. These proteasome inhibitors were found to considerably inhibit
ligand-stimulated degradation of the wild-type
-receptor; however,
their inhibitory effect was not observed when the cells expressing the
ubiquitination-deficient mutant
-receptor were analyzed. These
data suggest that the degradation process of the ligand-stimulated
-receptor involves the ubiquitin-proteasome proteolytic pathway.
Most receptors for polypeptide growth factors have a similar overall structural organization with an extracellular ligand-binding domain, a single transmembrane-spanning region, and an intracellular ligand-stimulatable tyrosine kinase domain. Based on the degree of structural similarities, the receptor tyrosine kinases can be divided into subfamilies(1) . Binding of the growth factor activates the intrinsic tyrosine kinase activity of the receptor, which leads to receptor autophosphorylation and to phosphorylation of intracellular substrates (for reviews, see (2) and (3) ). One role for receptor autophosphorylation is to present binding sites for signal transduction molecules containing one or two copies of a so-called Src homology 2 domain, which mediates the interaction with the receptor(4, 5) . Ligand binding is also accompanied by clustering of the receptors, and the receptor-ligand complex is ultimately delivered to and degraded in lysosomes.
Platelet-derived
growth factor (PDGF) ()promotes the growth of mesenchymal
cells in normal and pathological processes(6) . Two types of
the receptor for PDGF, designated
- and
-receptors, have been
identified(7, 8, 9) , and they both belong to
the receptor tyrosine kinase subfamily III(10) . We have
previously reported that the PDGF
-receptor undergoes
polyubiquitination as a consequence of ligand binding (11) and
have suggested that the ligand-induced ubiquitination plays a negative
regulatory role in mitogenic signaling of the PDGF
-receptor,
possibly by promoting the efficient degradation of the ligand-activated
receptor(12) . Ubiquitin is present in eukaryotes and is a
highly conserved 76-amino acid residue protein(13) . Evidence
supports the concept that ubiquitin conjugation to protein is
implicated in ATP-dependent proteolytic pathways for short-lived
proteins such as cyclins, Myc, Fos, and p53 (see (14) for a
review). A multisubunit 26 S (>2000 kDa) protease complex, which
specifically degrades proteins conjugated to ubiquitin, has previously
been described, and 20 S (
750 kDa) proteasome, also commonly known
as macropain or the multicatalytic proteinase complex, has subsequently
been shown to be the proteolytic core of the 26 S complex (for reviews,
see (15) and (16) ). The coupling of ubiquitin to
proteins is catalyzed by a family of small carrier proteins called E2s with or without the participation of the ubiquitin-protein
ligase, E3 (for reviews, see (17) and (18) ).
Recently, reagents that inhibit the ubiquitin-proteasome proteolytic
pathway in intact cells have become available, including
substrate-related peptidyl aldehydes (19, 20, 21) and a Streptomyces metabolite lactacystin(22) . In the present study, we
report that these proteasome inhibitors also considerably inhibit
ligand-stimulated degradation of the wild-type PDGF -receptor in vivo, and their inhibitory effect is lost when an
ubiquitination-deficient mutant
-receptor is analyzed, suggesting
that the degradation process of the ligand-stimulated PDGF
-receptor involves the ubiquitin-proteasome proteolytic pathway.
Each experiment presented in this study was repeated at least twice under identical conditions to confirm the reproducibility of the observations.
Ligand-stimulated degradation of the PDGF -receptor is
thought to play an important role in regulation of signal transduction
by the receptor. Therefore, it was of interest to clarify the mechanism
of the degradation process by analyzing effects of different protease
inhibitors.
First, we used substrate-related peptidyl aldehydes,
MG115 (19) and PSI (20) , as proteasome inhibitors and
E64-D as a calpain inhibitor and examined their effects on
ligand-stimulated degradation of the PDGF -receptor. PAE cells
expressing the wild-type PDGF
-receptor were metabolically labeled
for 2 h and then chased for 2 h with or without the different drugs.
Thereafter, the cells were stimulated with PDGF-BB for 0-120 min
at 37 °C in the presence of the respective drugs. After incubation,
the cells were lysed and immunoprecipitated with the
-receptor-specific antiserum PDGFR-3, and the immunoprecipitates
were analyzed by SDS-gel electrophoresis followed by fluorography. As
shown in Fig. 1, panel A, before stimulation of the
cells with PDGF-BB, the mature receptor band of 190 kDa was clearly
detected in each lane (lanes 1, 6, 11, and 16). After PDGF-BB stimulation, the intensity
of the band decreased rapidly in control (lanes 2-5) and
E64-D-treated (lanes 7-10) cells, whereas the intensity
decreased slowly in MG115- (lanes 12-15) and PSI-treated (lanes 17-20) cells. The efficiency of ligand-stimulated
degradation of the receptor was assessed by the rate of decrease in
intensity of the mature receptor band after PDGF-BB stimulation. As
shown in Fig. 1, panel B, quantitative analysis of the
gel revealed that, in control cells, the intensity of the band
decreased to 20% of the initial value after 30 min of PDGF-BB
stimulation and further decreased to an undetectable level after 90
min. Treatment of the cells with E64-D did not affect the rate of the
receptor degradation. On the other hand, both the treatment with MG115
and that with PSI similarly decreased the efficiency of the receptor
degradation; the intensity was approximately 50% after 30 min and was
still around 20% after 120 min. These results indicate that MG115- and
PSI-sensitive but E64-D-insensitive proteases are involved in the
degradation process of the ligand-stimulated PDGF
-receptor.
Figure 1:
Effects
of different protease inhibitors on ligand-stimulated degradation of
the wild-type PDGF -receptor. Panel A, PAE cells
expressing the wild-type human PDGF
-receptor were labeled for 2 h
at 37 °C with 100 µCi/ml EXPRE
S
S
[
S]protein labeling mix (DuPont NEN) and then
incubated for 2 h at 37 °C with 0.5% Me
SO (Control) (lanes 1-5) or different protease
inhibitors, 50 µM E64-D (lanes 6-10), 50
µM MG115 (lanes 11-15), and 50 µM PSI (lanes 16-20), in the presence of an excess of
unlabeled methionine and cysteine. Thereafter, the cells were incubated
with 100 ng/ml PDGF-BB at 37 °C in the presence of the respective
drugs for the indicated time periods. After incubation, the cells were
lysed, immunoprecipitated with PDGFR-3, and analyzed by SDS-gel
electrophoresis and fluorography. The migration position of the mature
form of the PDGF
-receptor (Rec) is indicated by an arrowhead. The relative migration positions of molecular mass
standards (myosin, 220 kDa; phosphorylase b, 97.4 kDa) run in
parallel are also indicated. Panel B, quantitation of the
mature form of the PDGF
-receptor on the gel shown in panel
A. Intensity of the mature receptor band in samples derived from
the cells treated with different protease inhibitors, E64-D (open
squares), MG115 (closed circles), and PSI (closed
squares), and from control cells (open circles) was
measured using the PhosphorImager with the ImageQuant software and is
expressed as percent of that at time zero.
Next, we examined individual as well as synergistic effects of
chloroquine and MG115 on ligand-stimulated degradation of the PDGF
-receptor. The wild-type receptor-expressing cells were treated
with these drugs one by one, or simultaneously, under the same
conditions as those described in the previous experiment. As shown in Fig. 2, treatment of the cells with MG115 reproducibly decreased
the efficiency of the receptor degradation. On the other hand,
chloroquine treatment did not appreciably affect the rate of the
receptor degradation. Furthermore, simultaneous treatment with
chloroquine and MG115 did not enhance the inhibitory effect of MG115 at
all. These results indicate that lysosomal proteolysis is not involved,
at least in the initial degradation step of the ligand-stimulated PDGF
-receptor.
Figure 2:
Effects of MG115 and chloroquine on
ligand-stimulated degradation of the wild-type PDGF -receptor. The
wild-type receptor-expressing cells were labeled as described in the
legend to Fig. 1and then chased for 2 h at 37 °C with 0.5%
Me
SO (control), 50 µM MG115, 100 µM chloroquine or both 50 µM MG115 and 100
µM chloroquine. Thereafter, the cells were incubated with
100 ng/ml PDGF-BB at 37 °C in the presence of the respective drugs
for the indicated time periods. After incubation, the cells were lysed,
immunoprecipitated with PDGFR-3, and analyzed by SDS-gel
electrophoresis and fluorography. Intensity of the mature receptor band
in samples derived from the cells treated with chloroquine (open
squares), MG115 (closed circles), or both MG115 and
chloroquine (closed squares), and from control cells (open
circles) was measured and is expressed as described in the legend
to Fig. 1.
In order to confirm the participation of proteasomes
in the degradation process of the ligand-stimulated PDGF
-receptor, we used another kind of proteasome inhibitor,
lactacystin, which is the most specific proteasome inhibitor available
to date(22) . We also examined the effect of another peptidyl
aldehyde, calpeptin, which is a specific calpain
inhibitor(23) . The wild-type receptor-expressing cells were
treated with these drugs under the same conditions as those described
in the previous experiment. As shown in Fig. 3, treatment of the
cells with lactacystin decreased the efficiency of the receptor
degradation, as expected. On the other hand, calpeptin treatment did
not affect the rate of the receptor degradation. These results,
together with the previous results using MG115 and PSI, strongly
support the interpretation that the proteasomedependent proteolytic
pathway is involved in the degradation process of the ligand-stimulated
PDGF
-receptor.
Figure 3:
Effects of calpeptin and lactacystin on
ligand-stimulated degradation of the wild-type PDGF -receptor. The
wild-type receptor-expressing cells were labeled as described in the
legend to Fig. 1and then chased for 2 h at 37 °C with 0.5%
Me
SO (control), 30 µM calpeptin, or 100
µM lactacystin. Thereafter, the cells were incubated with
100 ng/ml PDGF-BB at 37 °C in the presence of the respective drugs
for the indicated time periods. After incubation, the cells were lysed,
immunoprecipitated with PDGFR-3, and analyzed by SDS-gel
electrophoresis and fluorography. Intensity of the mature receptor band
in samples derived from the cells treated with lactacystin (closed
circles) or calpeptin (closed squares) and from control
cells (open circles) was measured and is expressed as
described in the legend to Fig. 1.
The wild-type PDGF -receptor undergoes
polyubiquitination as a consequence of ligand binding, and we have
found that a mutant PDGF
-receptor lacking the carboxyl-terminal
98 amino acid residues of the receptor (CT98 mutant) does not undergo
this posttranslational modification(11) . In order to evaluate
the role of the receptor ubiquitination on the proteasome-dependent
degradation process, we examined the effect of lactacystin on
ligand-stimulated degradation of the ubiquitination-deficient mutant
receptor. PAE cells expressing the wild-type or CT98 mutant receptors
were treated with lactacystin under the same conditions as those
described in the previous experiment. As shown in Fig. 4,
treatment of the wild-type receptor-expressing cells with lactacystin
reproducibly decreased the efficiency of the receptor degradation. On
the other hand, in the CT98 mutant receptor-expressing cells,
lactacystin treatment did not appreciably affect the rate of the
receptor degradation. Furthermore, even without lactacystin treatment,
the rate of degradation of the CT98 mutant receptor was lower compared
with that of the wild-type receptor, as reported
previously(11) ; the efficiency of receptor degradation in the
CT98 mutant receptor-expressing cells was nearly comparable with that
observed in the lactacystin-treated wild-type receptor-expressing
cells. These results indicate that the degradation process inhibitable
by lactacystin is dependent on ligand-induced ubiquitination of the
PDGF
-receptor.
Figure 4:
Effect of lactacystin on ligand-stimulated
degradation of the wild-type and CT98 mutant PDGF -receptors. PAE
cells expressing the wild-type or CT98 mutant PDGF
-receptors were
labeled as described in the legend to Fig. 1and then chased for
2 h at 37 °C with 0.5% Me
SO (control) or 100 µM lactacystin. Thereafter, the cells were incubated with 100 ng/ml
PDGF-BB at 37 °C in the presence of the respective drugs for the
indicated time periods. After incubation, the cells were lysed,
immunoprecipitated with PDGFR-3 for the wild-type and with PDGFR-HL2
for the CT98 mutant receptors, and analyzed by SDS-gel electrophoresis
and fluorography. Intensity of the mature receptor band in samples
derived from the cells treated with lactacystin (closed circles for the wild-type and closed squares for the CT98 mutant
receptors) and from control cells (open circles for the
wild-type and open squares for the CT98 mutant receptors) was
measured and is expressed as described in the legend to Fig. 1.
Ligand-induced endocytosis of the PDGF
-receptor precedes its intracellular degradation, and, hence, the
efficiency of internalization can affect that of degradation of the
receptor. Thus, it was necessary to examine effects of the drugs on
ligand-induced internalization of the receptor. The wild-type
receptor-expressing cells were preincubated for 2 h with chloroquine,
lactacystin, or calpeptin, then allowed to bind
I-PDGF-BB
for 1 h at 4 °C, washed, and further incubated with the respective
drugs at 37 °C for different time periods. After incubation and
subsequent acid wash at pH 3.7 to displace ligand bound to cell surface
receptors, the total and acid-nonreleasable (internalized)
cell-associated radioactivities were determined. Fig. 5shows
the ratio between the internalized and total cell-associated ligand
(expressed as percent radioactivity internalized). Internalization of
receptor-bound ligand occurred rapidly during the first 10 min and
further increased slowly for up to 30 min of incubation. Treatment of
the cells with chloroquine, lactacystin, and calpeptin did not
appreciably affect the internalization. These results clearly rule out
the possibility that the observed inhibitory effect of lactacystin on
the receptor degradation is due to a decrease in the internalization of
the PDGF
-receptor.
Figure 5:
Effects of different drugs on
internalization of I-PDGF-BB bound to PAE cells
expressing the wild-type PDGF
-receptor. The wild-type
receptor-expressing cells were incubated for 2 h at 37 °C with 0.5%
Me
SO (closed circles), 100 µM chloroquine (open circles), 100 µM lactacystin (closed squares), or 30 µM calpeptin (open squares). Thereafter, the cells were
cooled down on ice, incubated for 1 h at 4 °C with
I-PDGF-BB (
100,000 cpm/well), washed, and then
incubated at 37 °C for the indicated time periods in the continuous
presence of the respective drugs. The incubation was terminated by
removal of the medium, and the cells were incubated for 5 min on ice
with phosphate-buffered saline containing 1 mg/ml bovine serum albumin
or, alternatively, with the same buffer adjusted to pH 3.7 with acetic
acid (acid wash procedure). After the acid wash, the cells were lysed
to determine the amount of acid-nonreleasable (internalized) and total
cell-associated radioactivities. The rate of internalization is
expressed as the ratio between the internalized and total
cell-associated ligand (expressed as percent radioactivity
internalized). The standard deviation at each point was less than 5%
(triplicate determinations).
Finally, we also examined effects of chloroquine, lactacystin, and calpeptin on degradation of the receptor-bound ligand. The wild-type receptor-expressing cells were treated with these drugs under the same conditions as those described in the previous experiment. After incubation, the amount of trichloroacetic acid-nonprecipitable radioactivity in the medium, as a measure of degraded ligand, was recorded. Fig. 6shows the ratio between the degraded ligand and the initial cell-associated ligand (expressed as percent radioactivity degraded). Degradation of receptor-bound ligand increased rapidly in control cells, whereas the degradation increased somewhat slowly in lactacystin-treated cells. In calpeptin- and chloroquine-treated cells, appreciable degradation did not occur during 120 min of incubation. These results indicate that the degradation process of receptor-bound ligand is dependent completely on a chloroquine- and calpeptin-sensitive pathway as well as partially on a lactacystin-sensitive pathway.
Figure 6:
Effects of different drugs on degradation
of I-PDGF-BB bound to PAE cells expressing the wild-type
PDGF
-receptor. The wild-type receptor-expressing cells were
treated with 0.5% Me
SO (closed circles), 100
µM chloroquine (open circles), 100 µM lactacystin (closed squares) or 30 µM calpeptin (open squares), and incubated with
I-PDGF-BB under the same conditions as those described in
the legend to Fig. 5. After incubation, the medium was subjected
to trichloroacetic acid precipitation. The amount of trichloroacetic
acid-nonprecipitable radioactivity was taken as a measure of
degradation of
I-PDGF-BB. After removal of the medium,
the cells were lysed to determine cell-associated radioactivity. The
rate of degradation is expressed as the ratio between the degraded
ligand and the initial cell-associated ligand (expressed as percent
radioactivity degraded). The standard deviation at each point was less
than 6% (triplicate determinations).
Our present study demonstrates that, among different protease
inhibitors examined, including MG115, PSI, and lactacystin as
proteasome inhibitors, E64-D and calpeptin as calpain inhibitors, and
chloroquine as an inhibitor for lysosomal proteolysis, only the
proteasome inhibitors decrease the efficiency of ligand-stimulated
degradation of the wild-type PDGF -receptor. Furthermore, the
degradation process inhibitable by lactacystin is dependent on
ligand-induced ubiquitination of the receptor; the inhibitory effect of
lactacystin is lost when the ubiquitination-deficient mutant receptor
(CT98 mutant) is analyzed. These data indicate that the ligand-induced
ubiquitination and subsequent proteasome-dependent proteolysis are
involved in the degradation pathway of ligand-stimulated PDGF
-receptor.
The proteasome inhibitors used in the present study
could not completely block the receptor degradation. One possibility
that the dosage of the drugs was not enough to exert their full effects
is unlikely, since 200 µM MG115 gave the same result (data
not shown), and, more importantly, the rate of degradation of the
wild-type receptor in lactacystin-treated cells was nearly comparable
with that of the ubiquitination-deficient mutant receptor (CT98 mutant) (Fig. 4); the ubiquitin-proteasome proteolytic process must not
occur in these conditions. Another possibility is that the degradation
process of ligand-stimulated PDGF -receptor is catalyzed also by
some distinct protease(s), which is resistant not only to the
proteasome inhibitors but to the calpain inhibitors and chloroquine.
However, a more plausible explanation is that a fraction of accumulated
receptors after ligand-induced endocytosis might have become less
soluble, e.g. through association with the
detergent-insoluble cell fraction, which in our procedure was removed
by centrifugation from the lysate before immunoprecipitation (33) .
Our present observation that chloroquine treatment
did not affect the rate of the receptor degradation (Fig. 2)
does not necessarily exclude the possibility that lysosomal proteolysis
also contributes to the degradation of ligand-stimulated PDGF
-receptor. Because our current method records the fate of the
intact mature receptor molecule, that is to say the initial degradation
step of the receptor, partially cleaved receptor molecules, if any,
cannot be detected due to unpredictable changes in their molecular size
or immunological reactivity. It is rather conceivable that, following
the proteasome-dependent degradation of the receptor, the resultant
peptide fragments are delivered to and further degraded in lysosomes.
As shown in Fig. 6, the degradation of receptor-bound PDGF-BB was completely inhibited by chloroquine, as expected. The result fits the notion that the ligand-receptor complex is ultimately delivered to and degraded in lysosomes. Calpeptin was also found to completely block the ligand degradation, apparently suggesting the involvement of calpains in the degradation process. However, at present we attribute the effect of calpeptin to its possible inhibition of lysosomal cysteine proteases. On the other hand, lactacystin partially inhibited the ligand degradation. The result raises a possibility that the ubiquitin-proteasome proteolytic process functions also for the degradation of receptor-bound ligand upstream of the lysosomal pathway. The interpretation is further supported by our previous observation that the degradation of receptor-bound PDGF-BB by the cells expressing the ubiquitination-deficient mutant receptor (CT98 mutant) was about half that by the wild-type receptor-expressing cells(12) .
Taken together, our current hypothesis concerning the degradation
processes of the receptor-ligand complex of the PDGF -receptor is
as follows (see Fig. 7). After ligand stimulation, the receptor
is polyubiquitinated, and the internalized receptor-ligand complex is
initially degraded by the ubiquitin-proteasome proteolytic machinery.
Then the resultant peptide fragments are delivered to and further
degraded in lysosomes. The functional association of the two,
apparently distinct, proteolytic systems, the ubiquitin system and the
lysosomal autophagic system, has been described for the heat-induced
accelerated degradation of long lived proteins in the ts85 and the ts20
cells (which harbor a mutated thermolabile ubiquitin-activating enzyme, E1 (see (34) and (35) )). However, the
cooperative proteolysis by proteasome and lysosome, as suggested by the
present study, has not previously been reported. Further study is
necessary.
Figure 7:
Schematic illustration of possible modes
of ligand-stimulated degradation of the PDGF -receptor.
Ligand-activated receptor is polyubiquitinated, and then the
receptor-ligand complex is internalized and initially degraded by the
proteasome-dependent pathway. The resultant peptide fragments are
delivered to and further degraded in lysosomes. Ub stands for
ubiquitin conjugation.
In addition to the PDGF -receptor, other monomeric
receptors belonging to different kinds of the receptor tyrosine kinase
subfamily, such as the PDGF
-receptor, the epidermal growth factor
receptor, the colony-stimulating factor-1 receptor, the fibroblast
growth factor receptor-1(36) , and the c-kit-encoded
protein receptor (37) , have recently been found to be
polyubiquitinated after ligand stimulation. It is thus possible that
the novel mechanism for down-regulation of signal transduction by the
receptor, the ligand-induced receptor ubiquitination and its subsequent
proteosomal degradation, reported in the present study for the PDGF
-receptor is a general mechanism employed by most of the monomeric
receptor tyrosine kinases. Our future studies will be aimed at
exploring the possibility.