Department of Biochemical Sciences of the University of Florence, Italy
* Author for correspondence (e-mail: paola.chiarugi{at}unifi.it )
Accepted 27 February 2002
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Summary |
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Key words: Phosphotyrosine phosphatases, Tyrosine kinase receptor downregulation, Endocytosis, Ubiquitination, Redox regulation
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Introduction |
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c-Cbl, one of the numerous SH2-domain-containing proteins that bind to the
activated PDGF-r has been recently identified as a proteasomal E3-like
ubiquitin ligase involved in the downregulation of tyrosine kinase receptors
(Levkowitz et al., 2000).
C-Cbl ubiquitination is reported to target receptors for degradation by the
proteasome (Joazeiro et al.,
1999
; Bao et al.,
2000
). By this, cells became less sensitive to further PDGF
stimulation. Both ligand-induced internalisation and c-Cbl-mediated
ubiquitination require tyrosine phosphorylation of the receptor and can be
considered downregulation mechanisms that terminate receptor signalling
through degradation of the molecule
(Miyake et al., 1998
;
Levkowitz et al., 1999
).
The long-term downregulation response for PDGF-r signalling is essentially
mediated by messenger RNA expression reduction. It has been recently
demonstrated that the repression of pdgf-r mRNA level is mediated by
the c-Myc transcription early response gene. In particular, Myc-repression of
PDGF-r expression is responsible for the downregulation of the receptor during
the S and G2/M phase of the cell cycle
(Oster et al., 2000).
Downregulation of tyrosine kinase receptors by phosphotyrosine phosphatase
(PTPs) could be considered a simpler and faster way with respect to the
removal of the activable receptors from the membrane by clathrin-mediated
endocytosis and to the decrease of PDGF-r molecules by ubiquitin-mediated or
lysosomal proteolysis, or mRNA repression
(Östmann and Böhmer,
2001). Very little is known about the role of PTPs in PDGF-r
dephosphorylation, although many PTPs interact with this receptor. In
particular, the PTPs SHP-1 (Yu et al.,
1998
), SHP-2 (Qi et al.,
1999
), DEP-1 (Kovalenko et
al., 2000
), LMW-PTP (Chiarugi
et al., 1995
) and CD45 (Way
and Mooney, 1993
) have been described to dephosphorylate the
activated PDGF-r, although the real meaning of this dephosphorylation is
largely unclear. In fact, PTP action on tyrosine kinase receptors could lead
to a general attenuation of receptor signalling by targeting all receptor
phosphotyrosines or only the regulatory ones or, instead, by modulating
signalling through site-selective dephosphorylation. In this context, we would
like to elucidate the mechanisms of downregulation of tyrosine kinase
receptors that follows their agonist-induced activation. In particular, we
would like to compare the rate and the potency of each of the above mentioned
mechanisms.
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Materials and Methods |
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Cell culture and transfections
NIH-3T3 cells were routinely cultured in DMEM with the addition of 10%
fetal calf serum in 5% CO2 humidified atmosphere. All cell
treatments were performed at 37°C. 10 µg of pRcCMV-C12A-LMW-PTP was
transfected into NIH-3T3 cells using the calcium phosphate method
(Chiarugi et al., 1995). Stable
transfected clonal cell lines were isolated by selection with G418 (400
µg/ml). Mock-transfected cell lines were obtained by transfecting 2 µg
of pRcCMVneo alone. The clonal lines were screened for expression of the
transfected genes by (a) northern blot analysis and (b) ELISA using polyclonal
anti-LMW-PTP rabbit antibodies.
Immunoprecipitation and western blot analysis
1x106 cells were seeded in 10 cm plates in DMEM
supplemented with 10% FCS. Cells were serum starved for 24 hours before
receiving 30 ng/ml PDGF-BB. Cells were then lysed for 20 minutes on ice in 500
µl of RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS,
0.5% Na-Deoxicholate, 1% Nonidet P-40, 2 mM EGTA, 1 mM sodium orthovanadate, 1
mM phenyl-methanesulphonyl-fluoride, 10 µg/ml aprotinin and 10 µg/ml
leupeptin). Lysates were clarified by centrifugation and immunoprecipitated
for 4 hours at 4°C with 1-2 µg of the specific antibodies. Immune
complexes were collected on protein A Sepharose (Pharmacia), separated by
SDS/PAGE and transferred onto nitrocellulose (Sartorius). Immunoblots were
incubated in 3% bovine serum albumin, 10 mM Tris/HCl pH 7.5, 1 mM EDTA and
0.1% Tween-20 for 1 hour at room temperature, probed first with specific
antibodies and then with secondary antibodies conjugated with horseradish
peroxidase, washed and developed with the Enhanced Chemi-Luminescence kit.
Cell adhesion on fibronectin
1x106 cells were seeded in 10 cm plates in DMEM
supplemented with 10% FCS. Cells were serum starved for 24 hours before
detachment with 0.25% trypsin for 1 minute. Trypsin was blocked with 0.2 mg/ml
soybean trypsin inhibitor, centrifuged at 900 g for 10 minutes and
then re-suspended in 2 ml/10 cm dish of DMEM containing 0.2% BSA. Resuspended
cells were maintained in suspension with gentle agitation for 30 minutes at
37°C and then directly seeded in pre-coated dishes treated overnight with
10 µg/ml of human fibronectin in phosphate buffered saline (PBS), then
washed twice in PBS and blocked for two hours with 2% BSA in PBS.
PTP activity assay
The PTP activity was measured as previously reported
(Bucciantini et al., 1999).
Briefly, 1.5x106 cells were collected in 300 µl of 0.1 M
sodium acetate pH 5.5, 10 mM EDTA, 1 mM ß-mercaptoethanol then sonicated
for 10 seconds. The lysates were clarified by centrifugation, and 50 µl was
used in the PTP activity assays. We measured PTP activity either by using
p-nitrophenylphosphate or 32P-autophosphorylated PDGF-r. In the
first case, the cellular lysates were mixed with 250 µl of 10 mM
p-nitrophenylphosphate at 37°C for 30 minutes. The production of
p-nitrophenol was measured colorimetrically at 410 nm. The results were
normalised on the basis of total protein content. Quantification of PTP
activity using 32P-autophosphorylated PDGF-r as the substrate was
achieved by mixing the cellular lysates with equal amounts of
immunoprecipitated 32P-labeled PDGF-r and incubating at 37°C
for 15 minutes. Immunoprecipitated PDGF-r was labeled with
[
-32P]ATP (3000 Ci/mmol) in 100 µl of the kinase buffer
(see kinase activity assay) containing 100 nM PDGF-BB for 30 minutes at
30°C. The residual radioactivity on PDGF-r was assessed by SDS-PAGE
analysis. The autoradiogramm of the dried gel was analysed by densitometric
scanning (Chemi-Doc Biorad) and normalisation of the data was achieved by an
anti-PDGF-r immunoblot of the analysed samples.
Measurement of intracellular ROS
NIH3T3 cells were plated in 24-well plates (Corning) at a density of
2x104 cells per well in standard culture medium. 24 hours
later the cells were serum starved for an additional 24 hours. PDGF at 30
ng/ml was added for the indicated time followed by a 5 minute incubation with
20 µg/ml DCF-DA, an oxidation-sensitive fluorescent dye. Cells were then
detached from the substrate by trypsinisation and analysed immediately by flow
cytometry using a Becton Dickinson FACSCAN flow cytometer equipped with an
Argon laser lamp (FL-1; emission, 480 nm; band pass filter, 530 nm)
(Pani et al., 2000).
PDGF-r internalisation by trypsinisation
PDGF-r internalisation was determined by measuring the percentage of
receptors that were resistnat to trypsinisation according to the protocol of
Ceresa et al. (Ceresa et al.,
1998). Briefly, cells were serum starved for 24 hours and then
stimulated with 30 ng/ml of PDGF-BB at 37°C. The cells were washed twice
with PBS and incubated on ice for 8 minutes with ice-cold, 20 mM sodium
acetate, pH 3.7. The cells were washed with ice-cold PBS and incubated with
trypsin (1 mg/ml in PBS) in ice for 30 minutes with occasional rocking. The
reaction was stopped by addition of soybean trypsin inhibitor (1 mg/ml). Cells
were then washed and solubilised in lysis buffer for 10 minutes at 4°C.
The cell lysates were immunoprecipitated with anti-PDGF-r antibodies and
subjected to SDS-polyacrylamide gel electrophoresis (PAGE). The
trypsin-resistant 190 kDa PDGF-r is the endosomic receptor. Total PDGF-r
amount was evaluated in parallel in non-trypsinised cells and the ratio of the
two values has been calculated as 10%.
Inhibition of clathrin-mediated endocytosis
In order to inhibit PDGF-r endocytosis, NIH3T3 cells were incubated in
hypertonic medium (medium supplemented with sucrose 0.2 M) for 15 minutes at
37°C as reported previously (Lukacs et
al., 1997). Cells are then treated with PDGF-BB 30 ng/ml and
subjected to subsequent analysis.
PDGF-r kinase activity assay
Cell lysates in lysis buffer were subjected to immunoprecipitation with
anti PDGF-r subunit antibodies (Santacruz Biotec.). The immunoprecipitated
proteins were washed twice in lysis buffer and twice in 50 mM Tris-HCl, pH
7.4, containing 1 mM sodium orthovanadate and finally resuspended in 50 mM
Hepes at pH 7.4, 10 mM MnCl2. The reaction was started with the
addition of [32P]ATP (3000 Ci/mmol) (20 µCi) to all samples,
which were then incubated at 4°C for 10 minutes. The beads were then
washed once with 50 mM Tris-HCl, pH 7.4, and finally resuspended in 20 µl
of Laemmli's sample buffer, boiled for 5 minutes and separated by a 7.5%
SDS-PAGE (Sorkin et al.,
1993). The autoradiogram was scanned using Chemidoc Quantity One
software (Biorad). Normalisation was achieved by an anti-PDGF-r immunoblot
using a small amount of each of the analysed samples. In addition the PDGF-r
kinase activity was assayed using an exogenous substrate, glutathione
S-transferase-PLC-
(DeMali et al.,
1997
). Immunoprecipitates were incubated in the presence of 20 mM
PIPES, pH 7.0, 10 mM MnCl2, 20 µg/ml aprotinin and 10 µCi of
[
32-P]ATP (3000 Ci/mmol) for 10 minutes at 30°C in the
presence or absence of 0.5 µg of glutathione
S-transferase-PLC
1. The fusion protein included amino acid
residues 550-850 of rat PLC
1. The reaction was stopped by adding an
equal volume of 2xsample buffer (10 mM EDTA, 4% SDS, 5.6 mM
2-mercaptoethanol, 20% glycerol, 200 mM Tris-HCl, pH 6.8 and 1% bromphenol
blue). The samples were then incubated for 3 minutes at 95°C, spun and
resolved on 7.5% SDS-polyacrylamide gel electrophoresis, and the radiolabeled
proteins were detected by autoradiography. Normalisation was achieved by an
anti-PLC
1 immunoblot using a small amount of each of the analysed
samples.
Phosphatidylinositol 3-kinase assay
The PI3K assay was performed as described elsewhere
(Marra et al., 1995;
Chodhury et al., 1991
).
Briefly, serum-starved cells were incubated with 30 ng/ml of PDGF for 10
minutes and then lysed in RIPA buffer. Equal amounts of proteins were
immunoprecipitated using PY20 anti-phosphotyrosine antibody (Santacruz
Biotec.). After washing, the immunobeads were resuspended in 50 µl of 20 mM
Tris-HCl, pH 7.5, 100 mM NaCl and 0.5 mM EGTA. 0.5 µl of 20 mg/ml
phosphatidylinositol was added, mixed and incubated at 25°C for 10
minutes. 1 µl of 1 M MgCl2 and 10 µCi of
[
-32P]ATP (3000 Ci/mmol) were then added simultaneously and
incubated at 25°C for an additional 10 minutes. The reaction was stopped
by the addition of 150 µl of a mixture of chloroform, methanol and 37% HCl
(relative volumes 10:20:0.2). The samples were extracted with chloroform and
dired. Radioactive lipids were separated by thin-layer chromatography using
chloroform, methanol, 30% ammonium hydroxide and water (relative values
46:41:5:8). After drying, the plates were autoradiographed. The radioactive
spots corresponding to phosphatidylinositol phosphate were scraped and counted
in a liquid scintillation counter.
Cell cycle analysis
Cytofluorimetric analysis was performed according to Liu et al.
(Liu et al., 1995). Briefly,
2x105 cells were seeded into 6 cm dishes the day before
analysis to obtain exponentially growing cultures. Cells were rinsed twice
with cold PBS and harvested after trypsinization in 1 ml of 50 mg/liter
propidium iodide. Analysis was performed in a Becton Dickinson FACScan using
the Lysis II and Cell Fit cell analysis software according to the
manufacturer's procedure. Cell cycle rate evaluation was performed on five
independent dnLMW-PTP overexpressing clones (n=4 for each clone).
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Results |
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Furthermore, it has been reported that PDGF-r downregulation could be
achieved by proteasome-mediated degradation of the ubiquitinated receptor
(Miyake et al., 1998). We
analysed the time course of PDGF-r ubiquitination in PDGF-stimulated NIH3T3
cells and the results are shown in Fig.
1F. Again, PDGF-r labelling with ubiquitin follows a kinetic
pattern similar to its tyrosine phosphorylation, showing a maximum 10 minutes
after stimulation and slowly decreasing thereafter.
Furthermore, in order to study the fate of PDGF-r upon stimulation in NIH3T3 cells, we analysed the amount of total PDGF-r using a time course experiment with PDGF stimulation. The result (Fig. 2A) showed that the receptor level decreases by about 30% within 10 minutes of stimulation and then increases to 90% of the level seen in unstimulated cells (as indicated by densitometric analysis). Hence, it is likely that the decrease in the total amount of PDGF-r in Fig. 2A during agonist stimulation is only marginally caused by protein degradation. In any case the amount of downregulation of PDGF-r by physical removal from the cell surface and/or degradation is only a modest phenomenon. In order to confirm that the number of receptors that physically remain in the cell is relevant with respect to the amount of receptor eliminated by protein degradation, we analysed the ability of PDGF-r to be restimulated after a first 3 hour long stimulation and the removal of agonist by acid wash. The results (Fig. 2B) indicate that after the first treatment lasting 3 hours, PDGF-r is completely restimulated and shows a tyrosine phosphorylation level almost comparable with the original. A PDGF dose-response curve in NIH3T3 indicates that 3 ng/ml of the growth factor saturates the response. Using 30 ng/ml of PDGF in both treatments, we can exclude the possibility that the second increase in tyrosine phosphorylation of PDGF-r was caused by a limiting dose of PDGF during the first stimulation. Taken together these data suggest that the physical degradation of PDGF-r during agonist stimulation is only a marginal phenomenon and that the main part of the receptor is still able to be activated after the decrease in its tyrosine phosphorylation.
|
Relevance of different downregulation mechanisms in PDGF-r tyrosine
phosphorylation
Evaluation of the time course of each receptor downregulation mechanism
would allow us to discover which one is the most relevant for mitotic
regulation. Unfortunately, receptor dephosphorylation, receptor
internalisation and ubiquitination have a similar time course, each having a
maximum activity 10 minutes after stimulation and a reduced activity
thereafter. The use of specific inhibitors of each mechanism could allow us to
elucidate which is the most potent in terms of PDGF-r downstream signalling
inactivation. In particular, we used pervanadate in order to block PTP
activity in NIH3T3 cell upon PDGF treatment and MG-132 to selectively block
ubiquitinmediated protein degradation. The block in clathrin-mediated
endocytosis of PDGF-r was achieved by treatment with a hypertonic medium
containing 0.2 M sucrose (Lukaks et al., 1997). The tyrosine phosphorylation
level of total PDGF-r is shown in Fig.
3A; we demonstrate that only the block in dephosphorylation with
pervanadate leads to a long lasting hyperphosphorylation of the receptor. The
block in clathrin-mediated endocytosis leads to only a small delay in the
kinetic activity of receptor activation, but not an upregulation, whereas the
block in proteasomal degradation is ineffective in receptor switching off. On
the contrary, PTP inhibition by pervanadate does not affect the tyrosine
phosphorylation of the endosomic receptor
(Fig. 3B), which was analysed
after trypsin treatment. MG132 proteasome degradation inhibitor is efficient
in increasing the amount of ubiquitinated, but not degraded, PDGF-r.
(Fig. 3C, right panel).
Similarly, hypertonic medium pretreatment of cells almost completely blocks
PDGF-r ligand-induced internalisation, as shown by trypsin treatment
(Fig. 3C, left panel). The
inability of the block in receptor internalisation and proteasomal degradation
to affect the kinetics of PDGF-r activation suggests that amongst the
different downregulation mechanisms analysed the most significant is
phosphotyrosine dephosphorylation, although receptor internalisation retains a
minor role. In addition, it is likely that PTP dephosphorylation has
preferential membrane-associated PDGF-r pools as targets.
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Relevance of the different downregulation mechanisms in PDGF-r
signalling
By means of experiments similar to those described in the previous section,
we evaluated the activation of the main early signal transduction pathways
elicited by PDGF, namely the PI3K and the mitogen activated protein kinase
(MAPK) pathways, during selective blocking of the downregulation mechanisms of
PDGF-r. In particular, we were interested in clarifying the role of receptor
internalisation in MAPK activation. In fact there are conflicting data on this
point: receptor internalisation is generally considered to be a downregulation
mechanism for receptor signalling (Joly et
al., 1995), but the endosomic receptor seems to mainly mediate the
activation of Shc and Grb2 (Ceresa et al.,
1998
) and thus to have an intrinsic positive role in signal
transduction, at least for insulin receptor. We selectively blocked
ubiquitin-mediated degradation of PDGF-r (with MG132), receptor
internalisation (with hypertonic medium) or receptor dephosphorylation (with
sodium pervanadate). Lysates from these cells, stimulated or not with PDGF,
were used to evaluate MAPK activation (Fig.
4A). The results indicate that MAPK activation is considerably
reduced by the inhibition of PDGF-r internalisation, whereas it is unaffected
by the other treatments. Hence, we propose that neither receptor
ubiquitin-mediated degradation nor PDGF-r dephosphorylation are relevant for
MAPK activation, as none of the treatments has a positive effect on signalling
pathways. On the contrary, receptor internalisation seems to act positively on
MAPK activation pathway, as its inhibition by hypertonic medium leads to a
dramatic reduction in MAPK activation. In order to exclude the possibility
that 0.2 M sucrose suppresses MAPK pathway activation independently from
PDGF-r internalisation, we checked its specificity in inhibiting a PDGF-free
system, namely integrin-induced MAPK activation
(Fig. 4B). The results confirm
that the 0.2 treatment specifically inhibits PDGF-induced MAPK activation, and
it is particularly effective on integrin-induced MAPK activation. These data
on PDGF-r are in agreement with others for different growth factor receptors,
for which it has been demonstrated that the internalised receptor selectively
signals in the MAPK pathway (Ceresa et al.,
1998
; Chow et al.,
1998
).
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It has been demonstrated that PI3K plays a major role in initiating
PDGF-dependent DNA synthesis, whereas other signalling molecules are required
only for other PDGF-mediated functions
(Rosenkranz et al., 1999). We
selectively blocked all PDGF-r downregulation mechanisms, and the lysates from
PDGF-treated cells were used for the evaluation of PI3K activity
(Fig. 4C). Our data indicate
that only in cells treated with pervanadate and PDGF is there an increase in
PI3K activity with respect to cells treated with PDGF alone, whereas the other
treatments are almost ineffective. Taken together these findings show that the
main mitosis-correlated PDGF-induced signal transduction pathway, namely PI3K
activation, is influenced only by PTP inhibition. Hence, we propose again that
phosphotyrosine dephosphorylation is the most relevant signalling inhibition
system, awarding to receptor internalisation a specific and positive role in
MAPK signal transduction.
Correlation between PDGF-r tyrosine phosphorylation level and cell
cycle rate
In order to analyse the correlation between the tyrosine phosphorylation
level of PDGF-r and PDGF-r's main functional role, namely mitosis promotion,
we used a dominant-negative PTP to increase the tyrosine phosphorylation level
of the receptor. Dominant-negative PTPs are widely used to study the action of
these enzymes (Way and Mooney,
1993; Chiarugi et al.,
1995
; Kenner et al.,
1996
; Östmann and
Böhmer, 2001
) and commonly lead to an increase in tyrosine
phosphorylation of their PTP substrates. Many PTPs, such as LMW-PTP
(Chiarugi et al., 1995
), SHP-1
(Way and Mooney, 1993
), DEP-1
(Kovalenko et al., 2000
) and
others, act on phosphorylated PDGF-r, although in many cases a clear
specificity for dephosphorylation sites has not been demonstrated. In this
study, we use dominant-negative LMW-PTP (dnLMW-PTP), which we have already
reported to greatly increase the tyrosine phosphorylation level of PDGF-r
(Chiarugi et al., 1995
;
Chiarugi et al., 1998
). In
this work we exclusively use dnLMW-PTP, which is a useful tool to increase the
tyrosine phosphorylation level of the PDGF-r. Cells overexpressing dnLMW-PTP
were stimulated with PDGF, and the tyrosine phosphorylation level of PDGF-r
was evaluated in comparison with mock-transfected cells in a kinetic
experiment. The results, shown in Fig.
5A, again show that inhibition of PTP action on the activated
receptor leads to a long-lasting increase in its tyrosine phosphorylation, a
result comparable to that obtained with pervanadate
(Fig. 3A).
|
The relevance of dephosphorylation of PDGF-r for intracellular signalling pathways leading to G1 phase progression was confirmed by means of dnLMW-PTP overexpression. We evaluated the potency of PDGF signalling by cytofluorimetric analysis of cycling cells upon PDGF treatment during the block of each of the receptor downregulation mechanisms. NIH3T3 cells were serum starved for 24 hours and then stimulated with PDGF for 15, 17 and 20 hours. Unfortunately, treatment for a complete cell cycle with pervanadate, hypertonic medium and MG132 does not permit cell survival. The use of dnLMW-PTP allowed us to demonstrate that the difference in tyrosine phosphorylation level of PDGF-r in dnLMW-PTP-expressing cells and mock-transfected ones leads to a more rapid entry into G2/M phase, thus determining a decrease in cell cycle rate (Fig. 5B).
Taken together, these data suggest that the potency of the signal elicited by PDGF-r is proportional to its tyrosine phosphorylation level, which, in turn (Fig. 3A,B), is influenced mainly by PTPs and marginally by receptor internalisation but not by ubiquitination.
PDGF-r tyrosine phosphorylation regulation
The tyrosine phosphorylation level of PDGF-r during the 2 hour time course
may be regulated by the rate of phosphorylation and dephosphorylation. In this
light, we analyzed the kinase activity of PDGF-r and PTP activity during a 2
hour time course of PDGF stimulation. The results of an in vitro PDGF-r kinase
assay using immunoprecipitated PDGF-r from agonist-stimulated cells is
reported in Fig. 6A. The kinase
activity of PDGF-r shows a slow decrease during the first 2 hours, suggesting
that the rate of receptor activity does not remain constant over the 2 hour
stimulation with PDGF. Pervanadate treatment of NIH3T3 cells results in a
rapid upregulation of PDGF-r kinase activity 10 minutes after stimulation, but
not at longer times (Fig. 6A). PDGF-r kinase activity on an exogenous substrate such as PLC1 was
monitored during pervanadate treatment of cells
(Fig. 6B), and results in
agreement with the PDGF-r autophosphorylation assay were obtained. This
finding suggests a role for PTPs in the regulation of the receptor activity
only at very short times of agonist stimulation. Kazlauskas proposes that
although the kinase activity of the receptor is regulated within the first 10
minutes of stimulation, other receptor functions persist for longer times
(Bernard and Kazlauskas, 1999
).
We analysed the tyrosine phosphorylation level of PDGF-r Tyr-857 (by means of
anti-phospho-Y857-PDGF antibodies), which is required for full activation of
the receptor kinase activity (Baxter et
al., 1998
), in pervanadatetreated cells. The results, shown in
Fig. 6C, indicate that the
phosphorylation of this residue is extremely transient, reaching its maximum
after 10 minutes and completely disappearing thereafter. Hence, it is likely
that PTPs affect receptor kinase activity only after 10 minutes, because the
phosphorylation of the tyrosine residue required for activation of receptor
kinase activity is available only during the first 10 minutes.
|
PTP activity could be a housekeeping process or a finely regulated
phenomenon. In particular, the downregulation of the PDGF-r tyrosine
phosphorylation should caused by redox regulation of PTP activity during the
stimulation process (Östmann and
Böhmer, 2001; Chiarugi et
al., 2001
). First, we analyzed the PDGF-induced reactive oxygen
species (ROS) production in NIH3T3 cells
(Fig. 7A), which reaches its
maximum 10 minutes after stimulation and rapidly declines within 20 minutes.
These preliminary data are consistent with the current hypothesis of a redox
regulation of many PTPs by growth factor ROS production
(Östmann and Böhmer,
2001
). In this light, we analysed the PDGF-r tyrosine
phosphorylation level in a kinetic experiment during inhibition of ROS
production. For this purpose we treated cells either with catalase
(Fig. 7B), which reduces the
intracellular H2O2 concentration, or with diphenyliodide
(DPI) (Fig. 7C), a strong and
specific inhibitor of NADPH oxidase. The results indicate that the inhibition
of ROS production leads to a dramatic decrease in tyrosine phosphorylation of
PDGF-r. In addition, we checked that neither catalase nor DPI influenced the
tyrosine kinase activity of the receptor (data not shown). We further verified
that DPI and catalase treatments greatly reduced the redox inactivation of
PTPs during PDGF stimulation: for this purpose we use pNPP
(Fig. 7D) and an in vitro
phosphorylated PDGF-r (Fig. 7E)
as substrates. It is likely that the decreased ROS concentration in both
catalase and DPI-treated cells leads to a general upregulation of PTPs, which
are not downregulated via oxidation, thus remaining fully active on
phosphorylated PDGF-r.
|
On the basis of these data, we suggest that the tyrosine phosphorylation level of PDGF-r is regulated positively by the increase in its tyrosine kinase activity only during the first 10 minutes, and the activity rapidly decreasing thereafter. By contrast, PDGF-r phosphorylation level is negatively controlled by PTPs, which act on both its tyrosine kinase activity and on the general tyrosine phosphorylation level.
PDGF-r tyrosine phosphorylation is regulated in both of its distinct
phases of signaling
Recently, it has been demonstrated that prolonged and continuous exposure
to growth factors is required to commit cells to the cell cycle
(Jones and Kazlauskas, 2001).
In particular, the requirement for continuous exposure to mitogens can be
replaced by two short pulses of growth factor: one for 30 minutes at time zero
and the second at 8 hours. These extremely important data give rise to the
hypothesis that the PDGF receptor may remain tyrosine phosphorylated for a
very long time, as PDGF-activated signalling molecules continue to signal up
to 8 hours. This hypothesis is supported by the data of Bernard et al., which
demonstrated that the phosphorylation of the different tyrosines in PDGF-r is
temporal and spatial controlled (Bernard
and Kazlauskas, 1999
), thus suggesting that although the kinase
activity of the receptor is regulated within the first 10 minutes of
stimulation, other receptor functions, and in particular PI3K activation,
persist for longer times. We analysed the tyrosine phosphorylation level of
PDGF-r in NIH3T3 cells in a 0-9 hour time course experiment. The results,
reported in Fig. 8A, suggest
that PDGF-r remains tyrosine phosphorylated for up to 8 hours, although it
reaches its maximum level after 10 minutes and then remains at lower levels.
It is likely that although after a very short time the receptor becomes
phosphorylated at nearly all its tyrosines, fewer tyrosines remain
phosphorylated after a longer time, thus giving a general anti-phosphotyrosine
immunoblot signal lower than at 10 minutes.
|
In order to study PDGF-r downregulation during prolonged growth factor stimulation, we first analysed the amount of PDGF-r in a 0-9 hour time course experiment. Fig. 8B shows that the level of total PDGF-r decreases at 10 minutes, probably because of degradation, but then remains almost constant for the next 9 hours. These preliminary data suggest that the long-lasting downregulation of PDGF-r is not mediated by protein degradation either lysosomal or proteasomal. The role of PTPs in the long-lasting PDGF-r downregulation can be elucidated by pervanadate-treated cells or by dnLMW-PTP-expressing cells. Unfortunately 8-9 hours of pervanadate treatment is not compatible with cell life. By contrast, dnLMW-PTP expression gives rise to interesting results: the tyrosine phosphorylation level of PDGF-r is greatly influenced by LMW-PTP inhibition both at short and longer times (Fig. 9A), although it is more pronounced at the shortest times. We suggest that LMW-PTP acts on phosphorylated PDGF-r throughout the time that it remains phosphorylated and not only for the initial phase of signalling.
|
The main role of the PI3K pathway downstream of the second pulse (8 hours)
of PDGF stimulation has been unequivocally demonstrated by Jones et al.
(Jones et al., 1999). In fact,
synthetic PI3K lipid products can entirely and uniquely mimic the second pulse
of PDGF stimulation. To confirm our findings for the role PTP in the later
phase of signalling, we analysed the behaviour of the PI3K pathway throughout
a 9 hour time course of PDGF stimulation. We analysed the PI3K activity in a
time course experiment (Fig.
9B) in cells expressing dnLMW-PTP in comparison with
mock-transfected cells. The results reveal that PTP inhibition leads to
enhanced PI3K activity in both the immediate (10 minute) and the tardier (5-8
hours) waves of phosphoinositide production. Taken together, these findings
suggest that PTPs work throughout the period of PDGF-r phosphorylation,
probably acting on the specific residue/s that remains phosphorylated for up
to 8 hours.
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Discussion |
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The aim of this work was to compare the different downregulation mechanisms
acting on PDGF-r. In particular, we wanted to distinguish between the
efficiency of transient molecular modifications of PDGF-r leading to the
downregulation of membrane-bound receptors or the destruction of the PDGF-r
molecule by one of the possible proteolysis mechanisms. The data presented
herein, using tyrosine kinase receptors as a model, suggested that the
kinetics of tyrosine phosphorylation of PDGF-r are comparable with its
ligand-induced internalisation and with its ubiquitination
(Fig. 1A,C,D). The coexistence
of both the peak of activation of the signal (i.e. the tyrosine
phosphorylation of the PDGF-r) and of the maximum of endocytosis and
ubiquitination could support the hypothesis of a signalling role for both
mechanisms instead of them being termination systems. By contrast,
ligand-induced PDGF-r degradation, either obtained by clathrin-mediated
endocytosis and lysosomal delivery or ubiquitin-elicited proteasomal
proteolysis, is actually a system to limit the availability of transducing
receptors. Our data suggest that PDGF-r destruction is a marginal phenomenon
occurring either for a short time (Fig.
2A) or for longer, lasting growth factor stimulation
(Fig. 9B). We evaluated the
reduction in the amount of total PDGF-r during agonist treatment, and found it
to be reduced by 30%: this decrease could not account for the strong decline
in PDGF-r tyrosine phosphorylation (Fig.
1A) and of intracellular signalling pathway activation
(Fig. 3A,
Fig. 4A,B). In addition, we
demonstrated that the main part of the receptor remains available for further
stimulation (Fig. 2B). Our data
could not completely exclude a possible refilling of membrane-exposed
receptors with newly synthesised PDGF-r, thus contributing to the total amount
of PDGF-rs. It has been reported that upon agonist stimulation, PDGF-r
expression is repressed during the G1 phase by the c-Myc transcription factor
(Oster et al., 2000). For this
reason we suppose that receptor refilling could contribute only marginally to
receptor availability after agonist stimulation. The use of selective
inhibitors for anyone of the PDGF-r signalling termination systems point
towards PTPs as the main regulators of PDGF-r tyrosine phosphorylation level,
although they do not exclude a minor role for receptor endocytosis. Inhibition
of ubiquitination has almost no effect on PDGF-r tyrosine phosphorylation,
although blocking of clathrin-mediated endocytosis causes a small delay in
PDGF-r activation. Furthermore, our data suggest that ligand-induced PDGF-r
internalisation retains its role in MAPK pathway signal transduction. These
data are in agreement with previous reports on inhibition of clathrin-mediated
insulin receptor endocytosis (Ceresa et
al., 1998
), which demonstrate a selectivity in attenuation of Shc
tyrosine phosphorylation and MAPK activation. In addition, Burke has recently
demonstrated that epidermal growth factor signalling is regulated by
endocytosis and intracellular trafficking
(Burke et al., 2001
). Taken
together these data suggest that the modifications that PDGF-r undergoes
during agonist stimulation do not involve protein destruction (i.e. lysosome
delivery after internalisation or ubiquitin-mediated proteolysis is not
needed) but leave the receptor molecule at the plasma membrane available for
further activation.
Receptor tyrosine phosphorylation is a key event in the regulation of many
intracellular transduction pathways. SH2-containing proteins involved in
signal transduction are recruited to the plasma membrane through their
selective interaction with different phosphotyrosines in the activated
receptors. The inhibition of PTP action, either achieved with pervanadate
treatment (Fig. 3A) or with
dnLMW-PTP ectopic expression (Fig.
5A) causes an increase in the tyrosine phosphorylation of PDGF-r.
This increase causes an upregulation of the PI3K pathway but not of the MAPK
route and leads to an increase in G1 cell cycle phase rate
(Chiarugi et al., 1998). The
importance of the PI3K pathway for mitotic entry has already been reported by
Kazlauskas in 1999 (Rosenkranz et al.,
1999
). They showed that PDGF-dependent DNA synthesis was strictly
dependent on only one of the receptor elicited signalling routes, PI3K. Many
others enzymes are essentially required for other PDGF-mediated function, such
as chemotaxis. In this light, MAPK activation might depend on signalling from
the endosomic receptors, whereas PI3K recruitment and activation and
consequently mitotic entry, rely on tyrosine phosphorylation of plasma
membrane receptors.
The tyrosine phosphorylation level of a particular RTK is given by the
ratio between its intrinsic tyrosine kinase activity and the coordinated
activity of PTPs. The receptor tyrosine kinase activity is believed to be
controlled by agonist-induced dimerisation of the receptor and by the
transphosphorylation of a regulatory tyrosine, Tyr857, located in an
activation loop (Kazlauskas and Cooper,
1989). Recent data suggest that inhibition of PTP activity occurs
after RTK dimerization and thereby also contributes to receptor tyrosine
phosphorylation levels. Oxidation of the active site cysteine residue by
H2O2 has been identified as a mechanism for negative
regulation of PTPs (Lee et al.,
1998
). Interestingly, H2O2 is produced after
the stimulation of various RTK, such as PDGF-r, epidermal growth factor
receptor (EGF-r), insulin receptor and others
(Rhee, 2000
). Furthermore,
EGF-r stimulation is paralleled by a transient oxidation of PTP1B, and PDGF-r
is accompanied by a temporary oxidation of LMW-PTP
(Chiarugi et al., 2001
). Our
analysis of the tyrosine kinase activity of the receptor during inhibition of
PTPs by pervanadate suggests that PTPs affect the kinase activity of the
receptor only for the first 10 minutes, probably by dephosphorylating the
kinase loop tyrosine residue Tyr857. After this time, PTPs no longer influence
receptor tyrosine kinase activity, in agreement with previous reports that
suggest that Tyr857 is only briefly phosphorylated
(Bernard and Kazlauskas, 1999
;
Rosalind et al., 1996
).
Inhibition of the production of ROS in PDGF-stimulated cells, either achieved
by catalase pre-treatment or inhibition of the NADPH oxidase by DPI, leads to
the reduction of the potency of the PDGF-elicited signal, that is, a decrease
in the tyrosine phosphorylation level of the receptor. The effects of catalase
and DPI indicate that inhibition of PTPs has an important role in RTK
signalling and that the rescue of the catalytic activity of PTPs after
oxidation is followed by a dephosphorylation of the activated receptor, thus
terminating the signal. In view of this, we propose that during the first 10
minutes after stimulation the receptor tyrosine phosphorylation level
increases due to its intrinsic kinase activity and inhibition of PTPs via
oxidation. By contrast, during the second phase of the stimulation, the
phosphorylation level of the receptor declines as a function of the rescue
(via reduction) of PTP activity. This dephosphosphorylation closes the signal
elicited from the receptor.
Many studies have shown that in order to enter the S phase of the cell
cycle, a serum-arrested fibroblast must be continuously exposed to the growth
factor for about 2 hours (Westermark et
al., 1990). This means that the cell must remain in presence of
the growth factor long after all the signalling events correlated with mitosis
have occurred. In fact, some signalling enzymes are not only activated acutely
by exposure to growth factor but at a later time as well. Kazlauskas have
reported that both PI3K and protein kinase C show two waves of activation, one
within minutes of stimulation and one between 3 and 9 hours after the addition
of growth factors (Jones et al.,
1999
; Balciunaite et al.,
2000
). This late phase of enzymatic activity is required for cell
cycle progression. In this context, we suppose that PDGF-r could remain
phosphorylated for up to 9 hours in order to elicit both the first and the
second wave of enzyme activity. Our results show for the first time that the
tyrosine phosphorylation of PDGF-r is a long-lasting phenomenon that reaches a
maximum 10 minutes after the stimulation of the receptor, then slowly
decreases for 3-4 hours but remains at detectable levels for up to 9 hours.
Herein we show that PTPs act as terminators of the PDGF-r signal both in the
first phase of PDGF-r activation, when all tyrosine residues are
phosphorylated and recruit SH2-containing proteins, and in the second phase,
when only a few tyrosines remain phosphorylated and the long-lasting signals
take place. In fact, dnLMW-PTP expression causes an increase in both PDGF-r
short and long-lasting tyrosine phosphorylation and in PI3K first and second
activation waves. Kazlauskas demonstrated that the second phase of PDGF
required for S phase entry, at about 8 hours, could be mimicked by synthetic
PI3K products (Jones and Kazlauskas,
2001
) but not by other second messengers. Therefore, PTPs can act
on phosphorylated receptors when they are available, either minutes or several
hours after stimulation, thus permitting particular signals to last for a
longer time than others. These data are in agreement with a recent paper that
reports that for epidermal growth factor receptor the loss of tyrosine
dephosphorylation temporally precedes receptor degradation
(Burke et al., 2001
).
It is likely that PTPs affect RTK tyrosine phosphorylation in either a site-selective manner, that is, modulating preferentially some pathways rather than others, or causing a general phosphotyrosine decrease in content, thus causing a termination signal. Both these situations can be mediated by high site specificity of a particular PTP for the receptor, which acts in the first case on signalling phosphotyrosines or, in the second case, on the regulatory tyrosine in the activation loop of the receptor. Furthermore, our data indicate a preference of PTPs for membrane-exposed receptors, as we report that the endosomal receptor pool remains constantly phosphorylated for up to 2 hours (Fig. 1B-D), suggesting that PTPs may exclude this pool of receptors.
In conclusion we propose a model of PDGF-r downregulation in which the level of ligand-induced internalised tyrosine-phosphorylated receptors does not only play a termination role but also is a positive MAPK activation/transduction signal. PDGF-r protein destruction via lysosomal or ubiquitin-mediated proteolysis plays a marginal role, and the main part of the activated receptor is simply dephosphorylated by the concentrated action of PTPs. These enzymes, which are inhibited by the production of ROS owing to growth factor stimulation and reactivated thereafter by physiological thiols, are crucial for terminating the PDGF-r signal through a time-dependent dephosphorylation of available phosphotyrosines, thus contributing to the dynamic properties of PDGF-r signalling.
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---|
Baker, J. M., Khan, C. R., Cahill, D. A., Ullrich, A. and White,
M. F. (1990). Receptor mediated internalization of insulin
requires a 12-aminoacid sequence in the juxtamembrane region of the insulin
receptor beta-subunit. J. Biol. Chem.
265,16450
-16454.
Balciunaite, E., Jones, S., Toker, A. and Kazlauskas, A. (2000). PDGF initiates two distinct phases of protein kinase C activity that make an unequal contribution to the G0 to S transition. Curr. Biol. 10,261 -267.[Medline]
Bao, J., Alroy, I., Waterman, H., Schejter, E. D., Brodie, C.,
Gruenberg, J. and Yarden, Y. (2000). Threonine
phosphorylation diverts internalized epidermal growth factor receptors from a
degradative pathway to the recycling endosome. J. Biol.
Chem. 275,26178
-26186.
Baxter, R. M., Secrist, J. P., Vailancourt, R. R. and
Kazlauskas, A. (1998). Full activation of the
platelet-derived growth factor ß-receptor kinase involves multiple
events. J. Biol. Chem.
273,17050
-17055.
Bernard, A. and Kazlauskas, A. (1999). Phosphospecific antibodies reveal temporal regulation of platelet-derived growth factor-ß receptor signalling. Exp. Cell. Res. 253,704 -712.[Medline]
Bucciantini, M., Chiarugi, P., Cirri, P., Taddei, L., Stefani, M., Raugei, G., Nordlund, P. and Ramponi, G. (1999). The low Mr phosphotyrosine protein phosphatase behaves differently when phosphorylated at Tyr 131 or Tyr132 by Src kinase. FEBS Lett. 456, 73-78.[Medline]
Burke, P., Schooler, K. and Wiley, H. S.
(2001). Regulation of epidermal growth factor receptor signaling
by endocytosis and intracellular trafficking. Mol. Biol.
Cell 12,1897
-1910.
Ceresa, B. P., Kao, A. W., Santeler, S. R. and Pessin, J. E.
(1998). Inhibition of clathrin-mediated endocytosis selectively
attenuates specific insulin receptor signal transduction pathways.
Mol. Cell. Biol. 18,3862
-3870.
Chiarugi, P., Cirri, P., Raugei, G., Camici, G., Dolfi, F., Berti, A. and Ramponi, G. (1995). PDGF-receptor as a specific in vivo target for low M(r) phosphotyrosine protein phosphatase. FEBS Lett. 372,49 -53.[Medline]
Chiarugi, P., Cirri, P., Marra, F., Raugei, G., Fiaschi, T.,
Camici, G., Manao, G., Romanelli, R. G. and Ramponi, G.
(1998). The Src and signal transducers and activators of
transcription pathways as specific targets of low molecular weight
phosphotyrosine-protein-phosphatase in platelet-derived growth factor
signalling. J. Biol. Chem.
273,6776
-6785.
Chiarugi, P., Fiaschi, T., Taddei, M. L., Talini, D., Giannoni,
E., Raugei, G. and Ramponi, G. (2001). Two vicinal cysteines
confer a peculiar redox regulation to LMW-PTP in response to PDGF receptor
stimulation. J. Biol. Chem.
276,33478
-33487.
Chodhury, G. G., Wang, L. M., Pierce, J., Harvey, S. A. and
Sakaguchi, A. Y. (1991). A mutational analysis of
phosphatidylinositol 3-kinase activation by human colony-stimulating-factor-1
receptor. J. Biol. Chem.
266,8068
-8072.
Chow, J. C., Condorelli, G. and Smith, R. J.
(1998). Insulin-like growth factor-I receptor internalization
regulates signalling via the Shc/mitogen-activated protein kinase pathway, but
not the insulin receptor substrate-1 pathway. J. Biol.
Chem. 273,4672
-4680.
DeMali, K. A., Whiteford, C. C., Ulug, E. T. and Kazlauskas,
A. (1997). Platelet-derived growth factor-dependent cellular
transformation requires either phospholipase Cgamma or phosphatidylinositol 3
kinase. J. Biol. Chem.
272,9011
-9018.
Edinger, R. S., Rokaw, M. D. and Johnson, J. P. (1999). Vasopressin stimulates sodium transport in A6 cells via a phosphatidylinositide 3-kinase-dependent pathway. Am. J. Physiol. 277,575 -579.
Faure, R., Gaulin, G. F., Bourgoin S. and Fortier, S. (1999). Compartmentalization of the mitogen-activated protein kinase (MAPK) in hepatic endosomes: association with the internalized epidermal growth factor (EGF) receptor. Mol. Cell. Biol. Res. Commun. 1,132 -139.[Medline]
Flint, A. J., Tiganis, T., Barford, D. and Tonks, N. K.
(1997). Development of "subtrate trapping" mutants to
identify physiological substrates of protein tyrosine phosphatases.
Proc. Natl. Acad. Sci. USA
94,1680
-1685.
Heldin, C. H., Ostmann, A. and Ronnstrand, L. (1998). Signal transduction via platelet-derived growth factor receptors. Biochim. Biophys. Acta. 1378,F79 -F113.[Medline]
Joazeiro, C. A. P., Wing, S. S., Huang, H., Leverson, J. D.,
Hunter, T. and Liu, Y. (1999). The tyrosine kinase negative
regulator c-Cbl as a RING-type, E2-dependent ubiquitin protein ligase.
Science 286,309
-312.
Joly, M., Kazlauskas, A. and Corvera, S.
(1995). Phosphatidylinositol 3-kinase activity is required at a
postendocytic step in platelet derived growth factor receptor trafficking.
J. Biol. Chem. 270,13225
-13230.
Jones, S. M. and Kazlauskas, A. (2001). Growth-factor-dependent mitogenesis requires two distinct phases of signalling. Nat. Cell. Biol. 3, 165-172.[Medline]
Jones, S. M., Klinghoffer, R., Prestwich, G. D., Toker, A. and Kazlauskas, A. (1999). PDGF induces an early and a late wave of PI3-kinase activity, and only the late wave is required for progression through G1. Curr. Biol. 9, 512-521.[Medline]
Kazlauskas, A. and. Cooper, J. A. (1989). Autophosphorylation of the PDGF receptor in the kinase insert region regulates interaction with cell proteins. Cell. 58,1121 -1133.[Medline]
Kenner, K. A., Anyanwu, E., Olefsky, J. M. and Kusari, J.
(1996). Protein tyrosine-phosphatase 1-B is a negative regulator
of insulin and insulin-like-growth factor-1 stimulated signalling.
J. Biol. Chem. 271,19810
-19816.
Kovalenko, M., Denner, K., Sandström, J., Persson, C.,
Groß, S., Jandt, E., Vilella, R., Böhmer, F. and Östmann,
A. (2000). Site-selective dephosphorilation of the
platelet-derived growth factor ß-receptor by the receptor-like
protein-tyrosine phosphatase DEP-1. J. Biol. Chem.
275,16219
-16226.
Kranenburg, O., Verlaan, L. and Moolenaar, W. H.
(1999). Dynamin is required for the activation of
mitogen-activated protein (MAP) kinase by MAP kinase kinase. J.
Biol. Chem. 274,35301
-35304.
Langdon, W. Y., Hartley, J. W., Klinken, S. P., Ruscetti, S. K. and Morse, H. C. (1989). V-cbl an oncogene from a dual-recombinant murine retrovirus that induces early B-lineage lymphomas. Proc. Natl. Acad. Sci. USA 86,1168 -1172.[Abstract]
Lee, S. R., Kwon, K. S. and Kim, S. R. (1998).
Reversible inactivation of protein-tyrosine phosphatase 1-B in A431 cells
stimulated with epidhermal growth factor. J. Biol.
Chem. 273,15366
-15372.
Leof, E. B. (2000). Growth factor receptor signalling: location, location, location. Trends Cell Biol. 10,343 -348.[Medline]
Levkowitz, G., Waterman, H., Ettenberg, S. A., Katz, M., Tsygankov, A. Y., Alroy, I., Lavi, S., Iwai, K., Reiss, Y., Ciechanover, A. et al. (1999). Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1. Mol. Cell 4,1029 -1040.[Medline]
Levkowitz, G., Oved, S., Klapper, L. N., Harari, D., Lavi, S., Sela, M. and Yarden, Y. (2000). c-Cbl is a suppressor of the neu oncogene. J. Biol. Chem. 275,5532 -5539.
Liu, J. J., Chao, J. R., Jiang, M. C., Ng, S. Y., Yen, J. J. and Yang-Yen, H. F. (1995). Ras transformation results in an elevated level of cyclin D1 and accelleration of G1 progression in NIH-3T3 cells. Mol. Cell. Biol. 15,3654 -3663.[Abstract]
Lohse, M. J. (1993). Molecular mechanism of membran receptor desensitisation. Biochim. Biophys. Acta 1179,171 -188.[Medline]
Lukacs, G. L., Segal, G., Kartner, N., Grinstein, S. and Zhang, F. (1997). Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorilation. Biochem. J. 328,353 -361.[Medline]
Marra, F., Pinzani, M., De Franco, R., Laffi, G. and Gentilini, P. (1995). Involvement of phosphatidylinositol 3-kinase in the activation of extracellular signal regulated kinase by PDGF in hepatic stellate cells. FEBS Lett. 376,141 -145.[Medline]
Miyake, S., Lupher, M. L., Druker, B. and Band, H.
(1998). The tyrosine kinase regulator Cbl enhances the
ubiquination and degradation of the platelet-derived growth factor receptor
ß. Proc. Natl. Acad. Sci. USA
95,7927
-7932.
Miyake, S., Mullane-Robinson, K. P., Lill, N. L., Douillard, P.
and Band, H. (1999). Cbl-mediated negative regulation of
platelet-derived growth factor receptor-dependent cell proliferation. A
critical role for Cbl tyrosine kinase-binding domain. J. Biol.
Chem. 274,16619
-16628.
Mori, S., Rönnstrand, L., Claesson-Welsh, L. and Heldin, C.
H. (1994). A tyrosine residue in the juxtamembrane segment of
the platelet-derived growth factor ß-receptor is critical for
ligand-mediated endocytosis. J. Biol. Chem.
269,4917
-4921.
Oster, S., Marhin, W., Asker, C., Facchini, L. M., Dion, P. A.,
Funa, K., Post, M., Sedivy, J. M. and Penn, L. Z. (2000). Myc
is an essential negative regulator of platelet-derived growth factor beta
receptor expression. Mol. Cell. Biol.
20,6768
-6778.
Östmann, A. and Böhmer, F. (2001). Regulation of receptor tyrosine kinase signalling by protein tyrosine phosphatases. Trends Cell. Biol. 11,258 -265.[Medline]
Pani, G., Colavitti, R., Bedogni, B., Anzevino, R., Borrello, S.
and Galeotti, T. (2000). A redox signalling mechanism for
density-dependent inhibition of cell growth. J. Biol.
Chem. 275,38891
-38899.
Qi, J. H., Ito, N. and Claesson-Welsh, L.
(1999). Tyrosine phosphatase SHP-2 is involved in regulation of
platelet-derived growth factor-induced migration. J. Biol.
Chem. 274,14455
-14463.
Rhee, S. G. (2000). Hydrogen peroxide: a key messenger that modulates protein phosphorilation through cysteine oxidation. Science's STKE2000/53/pel (www.stke.sciencemag.org )
Rosalind, A., Segal, R. A., Bhattacharyya, A., Rua, L. A.,
Alberta, J. A., Stephens, R. M., Kaplan, D. R. and Stiles, C. D.
(1996). Differential utilization of Trk autophosphorylation
sites. J. Biol. Chem.
271,20175
-20181.
Rosenkranz, S., DeMali, C. A., Gelderloos, J. A., Bazenet, C. and Kazlauskas, A. (1999). Identification of the receptor-associated signalling enzymes that are required for platelet-derived growth factor-AA-dependent chemotaxis and DNA synthesis. J. Biol. Chem. 40,28335 -28343.
Shpetner, H., Joly, M., Hartley, D. and Corvera, S. (1996). Potential sites of PI-3 kinase function in the endocytic pathway revealed by the PI-3 kinase inhibitor, wortmannin. J. Cell Biol. 132,595 -605.[Abstract]
Sorkin, A., Ericksson, A., Heldin, C. H., Westermark, B. and Claesson-Welsh, L. (1993). Pool of ligand-bound platelet-derived growth factor ß-receptor remain activated and tyrosine phosphorilated after internalization. J. Cell. Physiol. 156,373 -382.[Medline]
Vieira, A. V., Lamaze, C. and Schmid, S. L.
(1996). Control of EGF-receptor signalling by clathrin-mediated
endocytosis. Science
274,2086
-2089.
Way, B. A. and Mooney, R. A. (1993). Activation
of phosphatidylinositol 3-kinase by platelet-derived growth factor and
insulin-like growth factor-1 is inhibited by a trans membrane phosphotyrosine
phosphatase. J. Biol. Chem.
268,26409
-26415.
Wells, A., Welsh, J. B., Lazar, C. S., Wiley, H. S., Gill, G. N. and Rosenfeld, M. G. (1990). Ligand-induced transformation by a noninternalizing epidermal growth factor receptor. Science 247,962 -964.[Medline]
Westermark, B., Siegbahn, A., Heldin, C. H. and Claesson-Welsh, L. (1990). B-type receptor for platelet-derived growth factor mediates a chemotactic response by means of ligand-induced activation of the receptor protein-tyrosine kinase. Proc. Natl. Acad. Sci. USA 87,128 -132.[Abstract]
Yoon, C. H., Lee, J. H., Jongeward, G. D. and Sternberg, P. W. (1995). Similarity of sli-1, a regulator of vulval development in C. elegans, to the mammalian proto-oncogene c-cbl. Science 269,1102 -1105.[Medline]
Yu, Z., Su, L., Hoglinger, O., Jaramillo, M. L., Banville, D.
and Shen, S. H. (1998). SHP-1 associates with both
platelet-derived growth factor receptor and the p85 subunit of
phosphatidylinositol 3-kinase. J. Biol. Chem.
273,3687
-3694.