From the
The transforming protein of simian sarcoma virus is homologous
to the platelet-derived growth factor (PDGF) B-chain. Fibroblasts
transformed with simian sarcoma virus constitutively produce a growth
factor that stimulates the endogenous tyrosine kinase of PDGF receptors
in an autocrine manner. Autophosphorylation of PDGF receptors upon
ligand stimulation provides binding sites for Src homology 2 domains of
intracellular signaling molecules, which thereby become activated. We
have characterized the PDGF receptor-mediated signal transduction in
NIH 3T3 cells transformed with a PDGF B-chain cDNA (Sis 3T3 cells) in
the absence and presence of suramin, a polyanionic compound that
quenches PDGF-induced mitogenicity and reverts the transformed
phenotype of the Sis 3T3 cells. Our data show that in the presence of
suramin the general level of tyrosine phosphorylation was decreased.
Nevertheless, autophosphorylated receptors complexed with substrates
persisted in the cells. Suramin had no effect on activation of
phosphatidylinositol 3`-kinase or on tyrosine phosphorylation of
phospholipase C-
PDGF
The sis oncogene of simian sarcoma virus (SSV) encodes a protein that is
homologous to the B-chain of PDGF
(3, 4, 5) .
SSV-transformed fibroblasts constitutively produce a growth factor that
stimulates endogenous PDGF receptors in an autocrine manner. A role for
PDGF in the autocrine stimulation of human tumors is suggested by the
coexpression of PDGF and PDGF receptors in gliomas
(6, 7) , sarcomas
(8) , and lung carcinomas
(9) , and by the recent finding that dominant-negative mutants
of PDGF as well as PDGF antibodies revert the transformed phenotype of
human astrocytoma
(10) and glioma cell lines
(11) . Only
cells that express PDGF receptor can be transformed by v- sis,
which suggests that the receptor is essential for transformation.
Upon binding of PDGF, the receptor undergoes dimerization, followed
by autophosphorylation of specific tyrosine residues in the
intracellular domain of the receptor. A number of autophosphorylation
sites have been identified in the intracellular domain of the PDGF
Suramin is a polyanionic compound that has been shown to quench
PDGF-induced mitogenicity and to revert the transformed phenotype of
SSV-transformed human foreskin fibroblasts. Suramin acts by inhibiting
binding of PDGF to its receptor and by dissociation of receptor-bound
PDGF
(22) . Previous studies have shown that suramin has no
effect on intracellular autophosphorylation of the receptor
(23, 24) .
In the present study, we have
characterized PDGF receptor-mediated signal transduction in NIH 3T3
cells transfected with the human B-chain cDNA. The transforming
potential of c-Sis/PDGF-BB is identical to that of v- sis and
the respective products are similarly assembled and processed
(25, 26, 27) . The impact of suramin, which
completely reverted the phenotype of the cells, on signaling via a
number of established signal transduction molecules was examined. In
the presence of suramin, the general level of tyrosine phosphorylation
was decreased. Nevertheless, complexes of autophosphorylated receptors
and signal transduction molecules persisted in the cell. To understand
the significance of the suramin-resistant receptor-substrate complexes,
we examined the state of tyrosine phosphorylation, kinase activity,
and/or complex formation of known substrates for the PDGF
The turnover rate of the PDGF receptors on
fibroblasts is increased in the presence of PDGF
(41) . The
increase in number of cell surface PDGF receptors in the
suramin-treated Sis 3T3 cells could therefore, at least in part, be due
to a disruption of ligand-dependent down-regulation of the receptors.
The results from pulse-chase analysis of
[
Shc is an SH2 domain-containing adaptor protein, known
to be involved in activation of Ras
(33) . To examine a possible
effect of suramin on tyrosine phosphorylation of Shc proteins (p46,
p52, and p66), the cell lysates were immunoprecipitated with
agarose-conjugated PY20 and then immunoblotted with anti-Shc
antibodies. PDGF-BB stimulation of NIH 3T3 cells induced a marked
increase in tyrosine phosphorylation of p46, p52, and, to a lesser
extent, p66. In Sis 3T3 cells, tyrosine-phosphorylated p46 and p52
proteins were readily detected. As shown in Fig. 5 C ( upper panel), there was a clear difference
between suramin-treated and untreated Sis 3T3 cells in the degree of
tyrosine phosphorylation of p52 and p46. Thus, suramin treatment
reduced the level of tyrosine-phosphorylated p52 to that seen in
unstimulated NIH 3T3 cells. The amount of immunoprecipitated Shc
proteins remained unchanged after suramin treatment as revealed by
immunoblotting using Shc antiserum (Fig. 5 C, lower
panel). We also performed the analysis by immunoprecipitating with
the Shc antibodies and blotting with phosphotyrosine antibodies. This
approach is less sensitive, since the Shc proteins co-migrate with the
heavy immunoglobulin chain. However, the same pattern of fluctuation of
the level of tyrosine-phosphorylated Shc was seen in the different
conditions (data not shown). Taken together, these data demonstrated
that tyrosine phosphorylation of Shc proteins (p46 and p52) was reduced
after suramin treatment, whereas the level of tyrosine phosphorylation
of PLC-
To show the
specificity of this experimental design, we used a GST fusion protein
containing the SH2 domain of p85 PI 3-K, in a parallel experiment. As
shown in Fig. 7 C, binding of the
This investigation is focused on the characteristics of PDGF
Analysis of the state of tyrosine phosphorylation of the PDGF
Analysis of tyrosine phosphorylation or state of activation of a
panel of known PDGF
Suramin treatment
also severely affected the activity state of Src members.
Autophosphorylation of Src, as well as the capacity of Src to
phosphorylate an exogenous substrate was below the level seen in
unstimulated NIH 3T3 cells. It has been reported that microinjection of
dominant-negative Fyn attenuates PDGF-induced DNA synthesis
(62) , indicating a role for Src members in mitogenic signaling.
It remains, however, to be shown whether or not Src family members
operate by converging into the Ras pathway, or if they act along a
parallel route.
The question whether the transforming signal in
sis-transformed cells is mediated by an intracellular pool of
ligand-activated PDGF receptors, or is generated at the cell surface by
an externalized ligand, has remained a controversial issue (reviewed in
Ref. 63). Our data provide evidence that some of the known PDGF
We thank Stuart A. Aaronson, Joop van Zoelen,
Alexander Sorkin and Graham Carpenter, Julian Downward, Tony Pawson,
Sara Courtneidge, Mike Waterfield, Joseph Schlessinger, Ben Margolis,
Ulf Rapp, and Susan Macdonald for their generous gift of reagents. For
advice and help with different assays, we thank Ann-Kristin Arvidsson,
Sven P, Koutaro Yokote, and Stefan Wennström. We also
gratefully acknowledge Annika Hermansson for technical assistance and
Ingegärd Schiller for secretarial assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and GTPase-activating protein of Ras. On the
other hand, kinase activation of Src and Raf-1, phosphorylation of
protein- tyrosine phosphatase 1D/Syp and Shc, and complex formation
with Grb2 were greatly diminished by suramin. A possible explanation
for our findings is that different PDGF receptor-coupled signaling
pathways are active in different structural or functional compartments
in the cell. Those pathways that are not affected by suramin might
elicit distinct cellular responses, which are not sufficient for growth
and transformation.
(
)
is a potent growth factor for cells
of mesenchymal origin. It is made up as a dimer of two related
polypeptide chains, designated A and B, which are assembled as homo- or
heterodimers (PDGF-AA, PDGF-BB, and PDGF-AB). PDGF has been implicated
as a regulator of cell proliferation during development and in wound
healing, but also in the stimulation of cell growth in pathological
conditions, e.g. atherosclerosis, tissue fibrosis, and
tumorigenesis
(1) . Two types of PDGF receptors, denoted
-
and
-receptors, which display differences in ligand binding
specificity, have been described
(2) . The B-subunit of PDGF
binds to both
- and
-receptors with high affinity, whereas
the A-subunit only binds to the
- receptor.
-receptor, and phosphorylation of these tyrosine residues allows
for specific interactions with SH2 domains of cytoplasmic signaling
proteins (reviewed in Ref. 12). When phosphorylated, Tyr-579 and
Tyr-581 in the juxtamembrane domain participate in binding of the
cytoplasmic tyrosine kinases Src, Fyn, and Yes
(13) . PLC-
binds to the region around phosphorylated Tyr-1009 and Tyr-1021 located
in the C-terminal tail
(14, 15, 16) ;
phosphorylated Tyr-1009 can also bind phosphotyrosine phosphatase
1D/Syp
(17) . The kinase insert contains several
autophosphorylation sites; two of these, Tyr-740 and Tyr-751, bind p85,
the regulatory subunit of PI 3-kinase
(18) , Tyr-751 is required
for binding of Nck
(19) , Tyr-771 binds the GTPase-activating
protein (GAP) of Ras
(20) , and Tyr-716 binds Grb2
(21) .
-receptor. Our data indicate that suramin treatment caused a
marked down-regulation of the activities of certain signal transduction
molecules, whereas others were less affected.
Cells and Tissue Culture
NIH 3T3 cells and Sis
3T3 cells (kindly provided by Stuart A. Aaronson) were cultured in
Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 10% fetal calf serum, 100 units/ml penicillin,
and 100 µg/ml streptomycin. Neomycin sulfate (0.1 mg/ml G418; Life
Technologies, Inc.) was included in the Sis 3T3 medium. 100
µM suramin (Germanin; Bayer) was added to the medium for
the 24 h preceding the experiment.
Antisera and Other Reagents
The rabbit antiserum
PDGFR-3 was generated using a synthetic peptide covering the murine
PDGF -receptor amino acids 981-994
(28) corresponding to amino acids 1013-1026 in the human
receptor; this antiserum reacts in a specific manner with the human
PDGF
-receptor
(29, 30) . The rabbit peptide
antiserum PDGFR-HL2 was provided by Joop van Zoelen, University of
Nijmegen, Toernooiveld
(31) . The mouse anti-phosphotyrosine
monoclonal antibody (PY20) was purchased from Transduction
Laboratories. The agarose-conjugated anti-phosphotyrosine (monoclonal
IgG2bk) was from Upstate Biotechnology Inc. Peroxidase-conjugated swine
anti-rabbit immunoglobulins was from DAKOPATTS AB (Copenhagen,
Denmark). Peroxidase-conjugated sheep anti-mouse immunoglobulin was
from Amersham Corp. The rabbit anti-PLC-
antiserum was a gift from
Alexander Sorkin and Graham Carpenter, Vanderbilt University,
Nashville, TN
(32) . The anti-Shc antiserum was provided by Tony
Pawson, Samuel Lunenfeld Research Institute, Mount Sinai Hospital,
Toronto
(33) . The monoclonal antibody used to immunoprecipitate
pp60
was mAb 327
(34) , a gift from Sara
Courtneidge, EMBL, Heidelberg. Rabbit antiserum against human GAP (PW
6.1) was provided by Julian Downward, Imperial Cancer Research Fund,
London. The rabbit polyclonal antiserum against c-Raf-1 (C-12) was from
Santa Cruz Biotechnology, Inc. The kinase-inactive mitogen-activated
protein kinase kinase (MEK) was a gift from Susan Macdonald, Onyx
Pharmaceuticals. The GST-SH2 Grb2 fusion protein was provided by Joseph
Schlessinger, NYU Medical Center, New York. The GST fusion protein
covering the SH2 domain of p85 of PI 3-K
(35) was a gift from
M. Waterfield, Ludwig Institute for Cancer Research, London. The GST
antibody was a gift from Ben Margolis, NYU Medical Center, New York.
The rabbit anti-PTP 1D/Syp was purchased from Upstate Biotechnology
Inc.
Confluent cells, grown in 12-well Linbro culture
dishes in the absence or presence of 100 µM suramin for 24
h, were washed once in Eagle's minimal essential medium,
supplemented with 20 mM Hepes, pH 7.4, and 1% newborn calf
serum. I-PDGF Binding
Assay
I-PDGF-BB was added to the medium (0.5 ml/well) to
a final concentration of 1 ng/ml and incubated for 1 h on ice. After
five washes with Eagle's minimal essential medium, 20 mM
Hepes, pH 7.4, 1% newborn calf serum, cell-bound radioactivity was
solubilized in 1% Triton X-100, 10% glycerol, 20 mM Hepes, pH
7.4, for 20 min and determined in a
-spectrometer. Nonspecific
binding was estimated from the extent of binding of
I-PDGF-BB in the presence of an 80-fold molar excess of
unlabeled PDGF. [
S]Methionine Labeling and
Immunoprecipitation-For pulse-chase analyses, cells in
25-cm
flasks were labeled in cysteine- and methionine-free
MCDB 104 medium supplemented with [
S]methionine
at 200 µCi/ml (Amersham; specific activity 3000 Ci/mmol) for 20 min
at 37 °C. The medium was aspirated, and an appropriate number of
cultures were rinsed with PBS and stored at
20 °C until
lysis. Dulbecco's modified Eagle's medium supplemented with
a 5-fold excess of methionine was added to the remaining cultures which
were incubated at 37 °C, for 30, 60, or 90 min. The monolayers were
then washed with ice-cold PBS and lysed in 0.5% Triton X-100, 0.5%
deoxycholate, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10
mM EDTA, 1% Trasylol (Bayer), 1 mM
phenylmethylsulfonyl fluoride (PMSF, Sigma). The lysates were clarified
by centrifugation at 10,000
g for 15 min and the
supernatants immunoprecipitated with antibodies specific for PDGF
-receptor (PDGFR-3) for 2 h at 4 °C. Protein A-Sepharose CL 4B
(Pharmacia LKB Biotechnology Inc.) was used to collect the immune
complexes. Precipitates were washed three times with lysis buffer and
once with ice-cold water and then eluted by heating the beads at 95
°C for 4 min in sample buffer containing 2% SDS, 0.2 M
Tris-HCl, pH 8.8, 0.5 M sucrose, 5 mM EDTA, 0.01%
bromphenol blue, and 2%
-mercaptoethanol (Carl Roth). After
separation by SDS-PAGE, using gels of 7.5% polyacrylamide, the gel was
soaked in Amplify (Amersham), dried, and exposed to Hyperfilm MP
(Amersham). [
P]Orthophosphate
Labeling-Confluent cells treated or not with 100
µM suramin for 24 h were rinsed three times in
phosphate-free Ham's F-12 medium supplemented with 10 mM
Hepes, pH 7.5, 0.1% fetal calf serum, and 50 µM
Na
VO
; 100 µM suramin was included
when appropriate. [
P]Orthophosphate (Amersham)
was added to a final concentration of 1 mCi/ml, and the cultures were
incubated at 37 °C for 3 h. One culture flask had been stimulated
overnight with 50 ng/ml PDGF-BB; for this culture, PDGF-BB was included
during the labeling period. After 3 h of labeling, the remaining
culture flasks were stimulated by incubation on ice and PDGF-BB was
added at 100 ng/ml; after incubation for 1 h, the cells were returned
to 37 °C for 7 min. Cells were then washed with ice-cold PBS and
solubilized in a lysis buffer consisting of 1% Triton X-100, 10%
glycerol, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1
mM PMSF, 1% Trasylol, 100 µM
Na
VO
. The cell lysates were centrifuged and
immunoprecipitated as described above for 2 h with PY20.
Immunoprecipitates were washed four times with lysis buffer and eluted
from the Sepharose beads as described above. Samples were subjected to
SDS-gel electrophoresis in a gradient gel of 7-12%
polyacrylamide, which after fixation was incubated in 10%
glutaraldehyde for 30 min in order to cross-link the proteins to the
gel matrix
(36) . The gel was then soaked in 1 M KOH at
55 °C for 1 h, to remove serine-bound phosphate
(37) , fixed
again, dried, and exposed to Hyperfilm MP (Amersham) using an
intensifying screen.
In Vitro Immune Complex Kinase Assay
Cells were
stimulated overnight with 50 ng/ml PDGF-BB or, alternatively, with 100
ng/ml PDGF-BB for 30 min on ice, followed by 7 min at 37 °C. After
washing with ice-cold buffer containing 20 mM Tris, pH 7.5,
150 mM NaCl, 100 µM NaVO
,
cells were solubilized in lysis buffer containing 1% Triton X-100, 10%
glycerol, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1
mM PMSF, 1% Trasylol, 100 µM
Na
VO
, 1 mM dithiothreitol (DTT). The
lysates were then centrifuged and the supernatants subjected to
immunoprecipitation with affinity purified HL-2 antibodies (specific
for the PDGF
-receptor; Ref. 31) for 2 h at 4 °C, followed by
incubation with protein A-Sepharose CL 4B. The beads were washed four
times with the lysis buffer and once with 20 mM Tris-HCl, pH
7.5, 150 mM NaCl, 100 µM
Na
VO
. Kinase assay was performed in 40 µl
of 20 mM Hepes, pH 7.4, 10 mM MnCl, 1 mM
DTT, containing 5 µCi [
-
P]ATP
(Amersham; specific activity 3000 Ci/mmol) for 10 min at 4 °C. The
kinase reaction was terminated by addition of 40 µl of 2
sample buffer for SDS-PAGE and heating at 95 °C for 4 min. Samples
were subjected to SDS-PAGE in a 7.5% polyacrylamide gel, which was
fixed, cross-linked, and treated with 1 M KOH, as described
above.
Src Kinase Assay
Cells were stimulated and
solubilized in the lysis buffer as described above for immune complex
kinase assay, but without DTT. Src protein was immunoprecipitated with
a monoclonal antibody, mAb 327. After washing, immune complex Src
kinase assay was performed as described above. As an exogenous
substrate for the Src kinase, acid-activated rabbit muscle enolase
(Sigma) was used, as described previously
(38) .
PI 3-Kinase Assay
Cells were stimulated as
described above, rinsed with PBS supplemented with 100 µM
NaVO
, and solubilized for 10 min on ice with 1%
Nonidet P-40, 10% glycerol, 20 mM Tris-HCl, pH 7.5, 150
mM NaCl, 100 µM Na
VO
, 1%
Trasylol, and 1 mM PMSF. Clarified cell lysates were
immunoprecipitated with PDGFR-3 and collected as described above. The
immobilized immune complexes were then washed three times with PBS
containing 1% Nonidet P-40, once with PBS, once with 0.1 M
Tris-HCl, pH 7.5, 0.5 M LiCl, once with distilled water, and
once with 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5
mM EDTA; all washes were at 0 °C. The beads were suspended
in 50 µl of 20 mM Tris-HCl, pH 7.5, 100 mM NaCl,
0.5 mM EGTA, and 0.2 mg/ml phosphatidylinositol (PI) (Sigma);
this solution had been sonicated for 15 min at 4 °C before use. A
preincubation was performed at room temperature for 10 min. Twenty
µCi of [
-
P]ATP and MgCl
(final concentration, 10 mM) were added and samples were
incubated for another 10 min. Reactions were stopped by addition of
chloroform, methanol, 11.6 M HCl (50:100:1), phospholipids
were extracted with chloroform and the organic phase washed with
methanol, 1 M HCl (1:1). Reaction products were concentrated
in vacuo, dissolved in chloroform, spotted on Silica Gel-60
plates (Merck) impregnated with 1% potassium oxalate and resolved by
chromatography in chloroform, methanol, 28% ammonia, water (43:38:5:7)
for 45 min. Phosphorylated products were detected by autoradiography on
RX films.
Raf-1 Kinase Assay
Cell lysates from Sis 3T3 and
NIH 3T3 were immunoprecipitated with 1.5 µg/ml Raf-1 polyclonal
antiserum (C-12). Immune complexes were washed three times in lysis
buffer and once in kinase buffer containing 30 mM Tris-HCl, pH
8.0, 20 mM MgCl, and 1 mM DTT, before
being resuspended in 30 µl of kinase assay mixture containing
kinase buffer, 0.5 µg of recombinant baculovirus-expressed
catalytically inactive MEK
(39) , 2 µM ATP, and 5
µCi of [
-
P]ATP/sample. Incubation was
for 30 min at 30 °C and was terminated by the addition of 40 µl
of hot 2
SDS-PAGE sample buffer, followed by incubation for 5
min at 95 °C. Samples were resolved by SDS-PAGE (7-12%
polyacrylamide gels), and gels were destained, dried, and subjected to
autoradiography.
Immunoblotting of PLC-
Cells were washed with PBS and lysed in a buffer containing
1% Triton X-100, 10% glycerol, 20 mM Tris-HCl, pH 7.5, 150
mM NaCl, 1 mM PMSF, 1% Trasylol, 100 µM
Na, GAP, Src, and
Shc
VO
. Immunoprecipitations were performed as
described above. The samples were separated by SDS-PAGE and
electrophoretically transferred to nitrocellulose membranes (Hybond C
extra; Amersham) in a buffer consisting of 20% methanol, 0.2 M
glycine, and 25 mM Tris-HCl, pH 8.3, at 400 mA for 3-4 h
at 4 °C. Blots were blocked by incubation in phosphate-buffered
saline containing 5% dried milk and 0.05% Tween 20 (Merck) for 1 h at
room temperature. Alternatively, the blots were blocked with
phosphate-buffered saline containing 5% bovine serum albumin and 0.05%
Tween 20 before probing with anti-phosphotyrosine antibody (PY20).
Blots were then incubated with different antisera. The blots were
washed three times for 10 min in PBS containing 0.05% Tween-20 and then
incubated with the peroxidase-conjugated swine anti-rabbit or sheep
anti-mouse immunoglobulins (1:5000 and 1:1000 dilutions, respectively).
After washing, bound antibodies were visualized using the ECL Western
blotting detection system (Amersham). The blots were reprobed after
removal of the first probe by incubation in 100 mM
-mercaptoethanol, 2% SDS, 20 mM Tris-HCl, pH 6.8, for 40
min at 60 °C, with occasional agitation.
Precipitation Using Gst-Grb2 and Gst-PI 3-K Fusion
Protein
Cultures of bacteria expressing GST-SH2 Grb2 or Gst-SH2
p85 PI 3-K were grown for 3 h at 37 °C in LB medium containing 0.5
mM isopropyl-1-thio--D-galactropyranoside.
Bacteria were collected by centrifugation, resuspended in 300
µl/sample 1% Triton X-100 in PBS, 2 mM EDTA, 0.1%
-mercaptoethanol, 0.2 mM PMSF, 1% Trasylol, and lysed by
sonication. The lysate was then clarified by centrifugation and the
supernatant was incubated with glutathione-Sepharose (600 µl of
packed beads) (Pharmacia) for 30 min at 4 °C. The
glutathione-Sepharose was washed three times with the lysis buffer and
then incubated with different cell lysates (70 µl/sample) for 2 h
at 4 °C. The samples were then washed five times with a lysis
buffer containing 1% Triton X-100, 10% glycerol, 20 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM PMSF, 1%
Trasylol, 100 µM Na
VO
and once
with ice-cold water, eluted and denatured by heating at 95 °C for 4
min in SDS-sample buffer, resolved on SDS-PAGE, and analyzed by
immunoblotting.
Characterization of the Effect of Suramin on
Sis-transformed NIH 3T3 Cells
We initially analyzed the effect
of suramin on NIH 3T3 cells transfected with the PDGF B-chain cDNA and
therefore expressing elevated levels of PDGF-BB (described in Ref. 24;
in this study denoted Sis 3T3 cells). In the absence of suramin, the
cells exhibited a transformed phenotype (Fig. 1 A,
left). In a previous study, the response of Sis 3T3 cells to
different concentrations of suramin were analyzed
(24) . In
agreement with their results, we found that treatment with 100
µM suramin for 24 h led to an apparently complete
reversion of the transformed phenotype (Fig. 1 A,
right). In the following experiments, cells treated with
suramin were examined microscopically before use to ensure that they
displayed the expected reverted morphology.
Figure 1:
Characterization of the effect of
suramin on Sis 3T3 cells. A, representative phase-contrast
microscopic fields of untreated Sis 3T3 cells ( left) and Sis
3T3 cells 24 h after the addition of 100 µM suramin
( right). B, extent of binding of
I-PDGF-BB (in counts/min/10
cells) to Sis 3T3
cells cultured with (+) or without (
) 100 µM
suramin, was determined in the absence ( filled bars)
or presence ( hatched bars) of an excess of unlabeled
PDGF-BB. C, Sis 3T3 cells, with (+) or without (
)
treatment with 100 µM suramin, were pulse-labeled for 20
min with [
S]methionine and chased for different
periods of time, as indicated. Immunoprecipitations were performed
using PDGFR-3. Samples were analyzed by SDS-PAGE and fluorography.
Molecular weight standards were run in parallel (not shown). The PDGF
-receptor precursor (160,000) and mature form (180,000), are
indicated.
Through its effect on
the interaction between PDGF and its receptors, suramin treatment leads
to an increased number of ligand-accessible cell surface PDGF receptors
on human fibroblasts transfected with v- sis (40) . This
was also the case for suramin-treated Sis 3T3 cells, as demonstrated by
use of a radioreceptor assay (Fig. 1 B); incubation of
suramin-treated Sis 3T3 cells with I-PDGF-BB revealed a
considerably increased number of specific ligand-accessible binding
sites, as compared to untreated cells. This result shows that suramin
treatment did not disrupt synthesis and transport of PDGF receptors to
the cell surface.
S]methionine-labeled Sis 3T3 cells
(Fig. 1 C) gave credit to this assumption, in agreement
with previous studies
(23, 42) . Thus, as a result of
suramin treatment, the mature form of the PDGF
-receptor
accumulated, whereas this form was hardly visible in the untreated
cells.
Analysis of Tyrosine-phosphorylated Proteins in
Suramin-treated and Untreated Sis 3T3 Cells by in Vivo
Labeling
In order to analyze PDGF receptor-mediated signal
transduction in Sis 3T3 cells, in the presence and absence of suramin,
[P]orthophosphate labeling of the cells was
performed, followed by immunoprecipitation with anti-phosphotyrosine
monoclonal antibodies (PY20) and SDS-PAGE analysis. Untransfected NIH
3T3 cells, stimulated with exogenous PDGF-BB for different time periods
were included in this experiment, for comparison. The duration of
stimulation with PDGF-BB was either 7 min or overnight; in the latter
case, the intention was to create a condition of ``chronic
stimulation'' more closely mimicking the situation in the Sis 3T3
cells. As seen in Fig. 2, the overall level of tyrosine
phosphorylation was decreased in the suramin-treated Sis 3T3 cells.
However, a component in the Sis 3T3 cells, which according to its
migration rate corresponded to the precursor form of the PDGF
-receptor, exhibited markedly preserved phosphorylation after
suramin treatment. Quantification showed that 66% of phosphorylated
precursor remained after suramin treatment, whereas the level of
phosphorylated mature receptors was reduced to 6% in the
suramin-treated cells, as compared to untreated cells. In addition, two
components of 74 and 54 kDa in the Sis 3T3 cells were largely resistant
to suramin treatment (indicated by arrows in Fig. 2).
These components could not be detected in the NIH 3T3 cells. A spectrum
of other molecules was phosphorylated after PDGF stimulation of NIH 3T3
cells, and similarly migrating molecules were seen in Sis 3T3 cells. It
is noteworthy that these components in general were of lower intensity,
both in the Sis 3T3 cells and in the long term stimulated NIH 3T3
cells, as compared to acutely stimulated NIH 3T3 cells. The resolution
of the autoradiogram shown does not allow a firm conclusion concerning
the status of these components in the suramin-treated Sis 3T3 cells.
However, longer exposures (not shown) indicated that tyrosine
phosphorylation of at least certain of the PDGF-induced substrates
persisted in Sis 3T3 cells treated with suramin.
Figure 2:
Analysis of tyrosine-phosphorylated
proteins by in vivo labeling. Confluent Sis 3T3 and NIH 3T3
cells, with (+) or without () 100 µM suramin
treatment for 24 h, were labeled with
[
P]orthophosphate for 3 h. The NIH 3T3 cells
were incubated in the absence (
) or presence (+) of 100
ng/ml PDGF-BB, for 30 min on ice followed by 7 min at 37 °C, or
alternatively, stimulated overnight with 50 ng/ml PDGF-BB
(++). Cell lysates were immunoprecipitated with PY20, and the
samples analyzed by SDS-PAGE and autoradiography. The relative
migration positions of molecular weight standards, run in parallel
(myosin, 200,000; phosphorylase b, 97,000; bovine serum
albumin, 69,000; ovalbumin, 46,000), are indicated to the left in the figure. Phosphorylated components present only in the Sis
3T3 cells irrespective of suramin treatment are indicated by
arrows. Migration positions of the mature and precursor forms
of the PDGF
-receptor are indicated by filled and
open diamonds, respectively. The lower part of the
figure shows a film exposed for a longer
time.
Analysis of PDGF Receptor-associated Components in
Suramin-treated and Untreated Sis 3T3 cells by in Vitro Complex Kinase
Assay
Signal transduction by tyrosine kinase growth factor
receptors is initiated by association between the receptors and
specific signal transduction molecules. We have previously employed
in vitro kinase assays, using immunoprecipitated receptor, to
show that the pattern of receptor-associated signal transduction
molecules is specific for different types of receptors in a highly
reproducible manner
(43) . In vitro kinase assays were
therefore performed on suramin-treated and untreated Sis 3T3 cells.
PDGF-BB-stimulated NIH 3T3 cells were included for comparison. The
cells were lysed and immunoprecipitated with an antiserum specific for
the PDGF -receptor (HL-2). The in vitro kinase assays
were performed on the immobilized immunoprecipitates, in the presence
of [
-
P]ATP, and the samples were analyzed
by SDS-PAGE, followed by autoradiography. Untreated Sis 3T3 cells and
PDGF-stimulated NIH 3T3 cells contained strongly labeled receptors and
a series of receptor-associated components. Based on their migration
rates, some of these components appeared to be present in both cell
types, whereas others were unique for each type, in agreement with the
data on in vivo
P-labeled cells (Fig. 2).
Fig. 3
shows that autophosphorylation of the mature form of the
PDGF
-receptor was markedly decreased after suramin treatment of
Sis 3T3 cells. However, a considerable degree of tyrosine
phosphorylation of the precursor (intracellular) form of the PDGF
-receptor remained in the presence of suramin, indicating
different effects of suramin on different cellular compartments. This
is in accordance with the data shown in Fig. 2. The effects of
suramin treatment on the extent of association between the receptor and
the different phosphorylated components, as compared to untreated Sis
3T3 cells, was assessed by use of a PhosphorImager instrument
(). Certain components remained in complex with the PDGF
-receptor to a considerable extent in the presence of suramin (see
components 5 and 7 in and Fig. 3). For other
components, however, the association was considerably reduced after
suramin treatment. For a group of components of about 70 kDa, the
association was almost abolished (see component 6 in and
Fig. 3
).
Figure 3:
Analysis of PDGF -receptor components
by in vitro complex kinase assay. Confluent Sis 3T3 cells and
NIH 3T3 fibroblasts, cultured with (+) or without (
) 100
µM suramin, were incubated in the absence (
) or
presence (+) of 100 ng/ml PDGF-BB for 30 min on ice, followed by 7
min at 37 °C. The lysates were immunoprecipitated using antiserum
specific for the PDGF
-receptor (HL-2). Kinase assays were
performed on the immune complexes, and samples were analyzed by
SDS-PAGE. The gel was treated with 1 M KOH at 55 °C for 1
h prior to autoradiography. Mature and precursor forms of the PDGF
-receptor are indicated by filled and open diamonds, respectively. The relative migration positions
of molecular weight standards, run in parallel (myosin, 200,000;
phosphorylase b, 97,000; bovine serum albumin, 69,000), are
indicated to the left. Components associating with the PDGF
-receptor in Sis 3T3 cells are numbered from top to bottom (see
Table I).
Analysis of Src Kinase Activity
We next examined
whether there were any differences between nontreated and
suramin-treated Sis 3T3 cells in the mode of interactions between the
PDGF -receptor and a number of proteins known to be involved in
PDGF-induced signal transduction. The activated PDGF
-receptor can
bind members of the Src family of tyrosine kinases and induce their
kinase activity
(44) . In order to examine the effect of suramin
on Src kinase activity, cells were cultured in the absence or presence
of 100 µM suramin for 24 h, lysed, and precipitated with a
Src-specific antibody (mAb 327). The specific kinase activity of Src
was then determined by using rabbit muscle enolase as an exogenous
substrate. As expected, PDGF-BB stimulation induced the kinase activity
of Src in NIH 3T3 cells (Fig. 4). The Src protein in untreated
Sis 3T3 cells exhibited a higher degree of kinase activity than that in
unstimulated NIH 3T3 cells, showing that Src is constitutively
activated in Sis 3T3 cells. Suramin treatment reduced the extent of
phosphorylation of enolase by Src in Sis 3T3 cells. The amount of Src
proteins remained unchanged after suramin treatment as revealed by
immunoblotting using Src antiserum (data not shown). Taken together
these data demonstrated that the constitutive Src kinase activity on
suramin-treated Sis 3T3 cells was reduced to a level below that seen in
unstimulated NIH 3T3 cells.
Figure 4:
Analysis of kinase activity of Src by
in vitro complex kinase assay. Confluent Sis 3T3 cells and NIH
3T3 cells, cultured in the absence () or presence (+) of
100 µM suramin were incubated with (+) or without
(
) 250 ng/ml PDGF-BB for 30 min on ice followed by 7 min at 37
°C. Alternatively, one culture was incubated overnight with 50
ng/ml PDGF-BB (++). The lysates were immunoprecipitated with
mAb 327 specific for Src. Kinase assays were performed on the immune
complexes with inclusion of rabbit muscle enolase, serving as an
exogenous substrate. Samples were analyzed by SDS-PAGE. The relative
intensity of enolase bands in the figure, as quantified by
densitometric scanning of the x-ray film, is indicated. Similar results
were obtained in three independent
experiments.
Analysis of Tyrosine Phosphorylation of PLC-
The effect of suramin on tyrosine
phosphorylation of PLC-,
RasGAP, and Shc
, RasGAP and Shc was investigated by
immunoblotting. PLC-
catalyzes the hydrolysis of
phophatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate
and diacylglycerol, leading to mobilization of Ca
and
activation of protein kinase C, respectively
(45) . Mutant PDGF
-receptors lacking the binding site for PLC-
are
mitogenically active
(15) , but PLC-
appears to affect
PDGF-induced mitogenic signaling under certain conditions
(46) .
In order to examine whether tyrosine phosphorylation of PLC-
was
affected by suramin, Sis 3T3 cells were cultured in the absence or
presence of suramin, lysed, and immunoprecipitated with anti-PLC-
antiserum. The immunoprecipitates were washed, separated by SDS-PAGE,
and transferred onto a nitrocellulose membrane. The blot was then
probed with an anti-phosphotyrosine monoclonal antibody (PY20). As
shown in Fig. 5 A ( upper panel),
tyrosine-phosphorylated PLC-
, as well as autophosphorylated PDGF
-receptors in complex with PLC-
, were readily detected in Sis
3T3 cells. Similarly, PDGF-BB stimulation of NIH 3T3 cells for
different time periods induced tyrosine phosphorylation of PLC-
,
as well as complex formation with the receptor. After exposure of the
Sis 3T3 cells to suramin for 24 h, tyrosine phosphorylation of
PLC-
and complex formation with the receptor remained virtually
intact. In addition, a component most probably corresponding to the
precursor form of the PDGF
-receptor coprecipitated with PLC-
after suramin treatment of the cells (Fig. 5 A, lane
2), indicating that binding between PLC-
and the precursor
form of the receptor occurs in intracellular compartments. Reprobing
the same filter with anti-PLC-
antiserum demonstrated that there
were similar amounts of immunoprecipitated PLC-
protein in the
samples as shown in Fig. 5 A ( lower panel).
Figure 5:
Tyrosine phosphorylation of PLC-,
RasGAP and Shc. A, confluent Sis 3T3 cells and NIH 3T3 cells,
cultured in the absence (
) or presence (+) of 100
µM suramin, were incubated with (+) or without
(
) 250 ng/ml PDGF-BB for 30 min on ice, followed by 7 min at 37
°C. Alternatively, one culture was incubated overnight with 50
ng/ml PDGF-BB (++). Immunoprecipitations were performed with
antiserum specific for PLC-
. The samples were resolved by
SDS-PAGE, transferred to nitrocellulose membrane and immunoblotted with
PY20 ( upper panel) and anti-PLC-
antiserum
( lower panel). Protein bands were visualized by
enhanced chemiluminescence (ECL). Migration positions of the mature and
precursor forms of the PDGF
-receptor are indicated by filled and open diamonds, respectively. B, as
in A, but immunoprecipitations were performed with anti-RasGAP
antiserum and blotting with PY20 ( upper panel) and
anti-RasGAP ( lower panel). The prominent band around
200 kDa in the upper panel has not been identified.
C, as in A, but immunoprecipitations were performed
with agarose-conjugated PY20 and blotting with anti-Shc antiserum
( upper panel). Alternatively, immunoprecipitations
were performed with anti-Shc antiserum and blotting with anti-Shc
antiserum ( lower panel). The relative migration
positions of relevant molecular weight standards, run in parallel
(myosin, 200,000;
-galactosidase, 116,500; ovalbumin, 46,000), are
indicated to the left in the panels. The relative intensity of
protein bands in the figure, as quantified by densitometric scanning of
the x-ray film, is indicated. Results similar to those shown in
panels A-C, were obtained in three or more independent
experiments.
In a parallel experiment, an equivalent set of
cell lysates were immunoprecipitated with anti-RasGAP antiserum
followed by immunoblotting with PY20. NIH 3T3 cells contained
tyrosine-phosphorylated RasGAP after PDGF-BB stimulation
(Fig. 5 B, upper panel).
Tyrosine-phosphorylated RasGAP could also be detected in Sis 3T3 cells,
to the same extent in the presence and absence of suramin. Subsequent
blotting with anti-RasGAP antiserum was performed to ensure that equal
amounts of material was loaded (Fig. 5 B, lower panel). That RasGAP tyrosine phosphorylation was
unaffected by suramin treatment of Sis 3T3 cells is in agreement with
data showing that mutant PDGF receptors, which lack the capacity to
bind and phosphorylate RasGAP, are still mitogenically active
(18) . Moreover, Ras activation appears to be controlled by the
activity of nucleotide exchange factors
(47, 48) (see
also below).
and RasGAP remained unchanged. These results are based on
three or more independent experiments, for each type of antisera,
followed by quantification of protein bands by scanning densitometry.
Analysis of PI 3-K Activity in Vitro
PI 3-K, which
phosphorylates the inositol ring of phosphatidylinositol in the D-3
position, has an important role in PDGF-induced motility responses
(49) ; in certain cell types, PI 3-K has also been implicated in
mitogenic signaling
(50, 51) . In order to examine
whether suramin treatment had any effect on PI 3-K activity, Sis 3T3
cells were cultured in the absence or presence of suramin, lysed, and
immunoprecipitated with antibodies against the PDGF- receptor
(PDGFR-3). The precipitates were subjected to a PI 3-K assay in the
presence of [
-
P]ATP and 0.2 mg/ml PI.
Phosphorylated lipids were extracted with chloroform and resolved by
thin layer chromatography. Phosphorylated PI in PDGF receptor
immunoprecipitates was formed as a result of PI 3-K activity, to very
similar extents in suramin-treated and untreated Sis 3T3 cells, as
shown in Fig. 6. Thus, the activity of PI 3-K was not affected by
suramin treatment. The receptor-associated PI 3-K activity in Sis 3T3
cells was twice that of unstimulated NIH 3T3 kept in serum, as averaged
from three independent experiments. Stimulation of NIH 3T3 cells with
PDGF-BB for 7 min at 37 °C led to a 9.5-fold increase in
receptor-associated PI 3-K activity, compared to unstimulated NIH 3T3
cells (average from three experiments).
Figure 6:
Phosphorylation of phosphatidylinositol by
PI 3-K. Confluent Sis 3T3 and NIH 3T3 cells, cultured in the absence
() or presence (+) of 100 µM suramin were
incubated with (+) or without (
) 250 ng/ml PDGF-BB for 30
min on ice, followed by 7 min at 37 °C. Immunoprecipitations were
performed with PDGFR-3. Immune complexes were subjected to PI 3-K assay
and the PI 3-K reaction products were analyzed by thin layer
chromatography and autoradiography. The position of
phosphatidylinositol phosphate ( PIP) and the origin
( ORI) are indicated. Quantification of the
phosphatidylinositol phosphate spots were made using scanning
densitometry and are presented as relative intensity in the lower part
of the figure.
Tyrosine Phosphorylation of Grb2-associated
Proteins
The role of Ras activation in the signaling of growth
factor receptors has recently been elucidated. Nucleotide exchange on
Ras is mediated by Sos1, which through its binding to Grb2 becomes
associated with autophosphorylated tyrosine residues on the growth
factor receptor itself, or on downstream adaptor molecules
(48) . Complex formation between the PDGF -receptor and
Grb2 has been described in NIH 3T3 cells
(21) ; however, with
the available reagents, the complex was detected only with very low
efficiency (data not shown). Therefore, to examine whether association
between the PDGF receptor and Grb2 occurs in Sis 3T3 cells, a GST-SH2
Grb2 fusion protein was used. Cells were grown in the absence or
presence of suramin, lysed, and the lysates were incubated with the
fusion protein immobilized on glutathione-Sepharose 4B. Components
associating with the Grb2 SH2 domain were separated by SDS-PAGE and
transferred onto nitrocellulose membranes, which were probed with
anti-phosphotyrosine antibodies (PY20). In stimulated NIH 3T3 cells,
tyrosine-phosphorylated PDGF receptors were in complex with the
immobilized Grb2 fusion protein (Fig. 7 A). In addition,
two tyrosine-phosphorylated components of about 70 kDa were detected.
One band appeared only after stimulation with PDGF-BB, whereas the
other band was detected independently of stimulation of NIH 3T3 cells.
In untreated Sis 3T3 cells, tyrosine-phosphorylated PDGF receptors
associated with the Grb2 fusion protein as well as the two
tyrosine-phosphorylated 70-kDa components. In the presence of suramin,
the receptor band and the two 70-kDa components were hardly detectable.
It is noteworthy that components around 70 kDa were no longer
phosphorylated in the suramin-treated Sis 3T3 cells analyzed by in
vitro kinase assay (see Fig. 3and ). Equal
amounts of the Grb2 fusion protein were present in each sample, as seen
from probing the filter with anti-GST antibodies (data not shown). In
addition to the receptor and the 70-kDa component(s), Shc was found in
complex with the Grb2 SH2 domain. In the sample from acutely stimulated
NIH 3T3 cells, p52 was visualized by blotting with phosphotyrosine
antiserum (Fig. 7 A). Blotting of the filter in
Fig. 7A with anti-Shc antiserum showed that p46 and p52
also formed a complex with Grb2 SH2 in the sample from untreated Sis
3T3 cells. In suramin-treated cells the Shc immunoreactivity was
abolished (data not shown; compare Fig. 5 C, upper panel).
Figure 7:
Analysis of Grb2 associated proteins.
Confluent Sis 3T3 cells and NIH 3T3 cells cultured in the absence
() or presence (+) of 100 µM suramin, were
incubated with (+) or without (
) 250 ng/ml PDGF-BB for 30
min on ice followed by 7 min at 37 °C. One culture was incubated
overnight with 50 ng/ml PDGF-BB (++). Cell lysates were
incubated with GST-SH2-Grb2 immobilized on glutathione-Sepharose 4B,
the samples resolved by SDS-PAGE and immunoblotted with PY20
( A) and anti-PTP1D/Syp ( B).The relative intensity of
protein bands in the figure, quantified by scanning densitometry of the
x-ray film, is indicated in the lower part of the figure. Similar
results were obtained in at least three independent experiments.
C, as in A, but cell lysates were incubated with
GST-SH2-p85 of PI 3-K and immunoblotting was performed with PY20. The
relative migration positions of molecular weight standards, run in
parallel (myosin, 200,000;
-galactosidase, 116,500; bovine serum
albumin, 81,000; Bio-Rad SDS-PAGE standards), are indicated to the
left.
To explore the possibility that the 70-kDa
component(s) seen in both Sis 3T3 and NIH 3T3 cells corresponded to the
SH2 domain-containing phosphatase PTP 1D/Syp
(52, 53, 54) , which recently was shown to bind
the SH2 domain of Grb2
(55) , the filter shown in
Fig. 7A was stripped and reprobed with anti-Syp
antibodies. As shown in Fig. 7 B, two components were
detected, whose migration rates exactly matched those of the 70-kDa
tyrosine-phosphorylated components visualized in
Fig. 7A. In NIH 3T3 cells, the lower band appeared only
after stimulation, whereas the upper band was detected in unstimulated
cells as well. In Sis 3T3 cells, the Syp antiserum visualized two
components, which both disappeared after suramin treatment. These
observations are consistent with the conclusion that the 70-kDa
components, which bind less efficiently to Grb2 SH2 after suramin
treatment of the cells, are related to PTP 1D/Syp.
-receptor from Sis
3T3 cells to the GST SH2 p85 was unaffected by suramin. This is in
agreement with the result in Fig. 6, where there was no effect of
suramin treatment on the level of PI 3-K activity in Sis 3T3 cells.
Analysis of Raf-1 Kinase Activity
Raf-1 is
activated by phosphorylation on serine in response to a wide variety of
mitogens
(56) . It seems likely that Raf-1 is a component of
many mitogenic signaling pathways involving different classes of
receptors. One, possibly major, such pathway is routed via Ras, which
in its GTP-bound form exists in complex with Raf-1
(57) . Raf-1
has been found to phosphorylate and activate mitogen-activated protein
kinase kinase MEK, an activator of MAP kinase (for a review, see Ref.
58), thus linking the Raf-1 signaling pathway with that of MAP kinase.
We used a Raf kinase assay to examine the activity of Raf-1 in the
absence or presence of suramin in the medium. Proteins from cell
lysates were immunoprecipitated with antiserum specific to Raf-1 (C-12)
and assayed for kinase activity measuring the phosphorylation of
recombinant, catalytically inactive MEK. The data in Fig. 8show
that MEK was phosphorylated in Sis 3T3 cells in the absence of suramin
and in PDGF-stimulated NIH 3T3 cells. Fig. 8also demonstrates
that suramin treatment of Sis 3T3 cells was accompanied by a decrease
in phosphorylation of MEK to the level seen in unstimulated NIH 3T3
cells, indicating that the constitutive kinase activity of Raf-1 was
reduced after suramin treatment of Sis 3T3 cells. Similarly, suramin
changed the phosphorylation state of Raf-1 from phosphorylated form to
an unphosphorylated form, seen as a shift in electrophoretic mobility
of Raf-1 in SDS-gel electrophoresis (data not shown).
Figure 8:
Analysis of kinase activity of Raf-1 by
in vitro complex kinase assay. Confluent Sis 3T3 cells and NIH
3T3 cells cultured in the absence () or presence (+) of 100
µM suramin were incubated with (+) or without
(
) 250 ng/ml PDGF-BB for 30 min on ice, followed by 7 min at 37
°C. One culture was incubated overnight with 50 ng/ml PDGF-BB
(++). Cell lysates were immunoprecipitated with an antibody
specific for Raf-1 and assayed for phosphorylation of catalytically
inactive MEK in the immune complex. Samples were resolved by SDS-PAGE
and detected by autoradiography. The relative intensity of MEK bands in
the figure, quantified by scanning densitometry of the x-ray film, is
indicated. Similar results were obtained in three independent
experiments.
-receptor-mediated signal transduction in NIH 3T3 cells
transformed with a PDGF B-chain cDNA (Sis 3T3 cells). We and others
have shown that the transformed phenotype of such cells can be reverted
by treatment with the polyanionic drug suramin; in the present study we
examined the consequences of suramin-induced reversion for signaling
via a number of known signal transduction molecules. Src, PI 3-K,
RasGAP, PLC-
, PTP 1D, Grb2, Shc, and Raf were characterized with
regard to complex formation with the receptor, tyrosine
phosphorylation, or state of activation. Our data indicate that all of
these molecules may participate in autocrine signal transduction in Sis
3T3 cells. This view is based on a comparison of Sis 3T3 cells with
untransfected NIH 3T3 cells and NIH 3T3 cells stimulated with PDGF-BB,
either for a few minutes or overnight. As expected, the levels of
tyrosine phosphorylation/activation of these substrates in the Sis 3T3
cells were in several cases (see Figs. 4 and 5, A and
C) more similar to those in long term stimulated NIH 3T3 cells
than in acutely stimulated cells; apparently, long term activation of
the PDGF
-receptor leads to down-regulation not only of the
receptor, but also of several of the downstream effector molecules.
-receptor in vivo and in vitro showed that
activated, autophosphorylated receptors remain in complexes with signal
transduction molecules in suramin-treated cells. Although the general
level of phosphorylation was decreased, the decrease was more
pronounced for some components than for others. Thus, the mature form
of the PDGF
-receptor was dephosphorylated more efficiently than
the precursor form. Similar findings have been reported by other
investigators
(24, 59) . Our data therefore strongly
support the notion that suramin preferentially affects ligand-receptor
complexes at the cell surface, leaving intracellular complexes intact.
This is in agreement with previous studies
(23, 24) .
-receptor signaling molecules showed that
these were not equally affected by suramin treatment. Whereas the level
of tyrosine phosphorylation of GAP and PLC-
and the activity state
of PI 3-K remained unchanged in suramin-treated Sis 3T3 cells as
compared to untreated cells, our data indicated that the Ras activation
pathway in Sis 3T3 cells was affected by suramin. Recent studies have
identified Ras as a switchboard for several types of growth stimuli,
transducing them to signals along a pathway that eventually results in
cell division
(60) . Our study has highlighted the role of the
Ras pathway in autocrine transformation, mediated by an activated PDGF
-receptor. Thus, after suramin treatment of Sis 3T3 cells, the
PDGF
-receptors no longer associated with a Grb2-SH2 fusion
protein. Our interpretation of these data is that the interaction
between the PDGF
-receptor and the SH2 domain of Grb2 occurs
within the suramin-sensitive pool of PDGF receptor-ligand complexes,
i.e. those that are located at the cell surface. A fusion
protein comprising the SH2 domain of p85 of PI 3-K, on the other hand,
still bound PDGF
-receptors from suramin-treated Sis 3T3 cells. We
argue that complexes between the
-receptor and p85 were located in
a suramin-inaccessible cellular compartment. The cellular compartment
containing suramin-resistant receptor complexes may include the
endoplasmic reticulum, since the receptor precursor appeared to be in
complex with PLC-
(see Fig. 5 A). We further found
that tyrosine-phosphorylated components of 52 and 70 kDa had lost their
association with Grb2, or with the receptor, as a result of suramin
treatment. The 70-kDa component was found to be immunologically related
and possibly identical to PTP 1D (Syp), the role of which in signal
transduction is not clear. The 52-kDa component was identified by
immunoblotting as p52, which is known to form complex with Grb2
(33) . In a separate experiment, we showed that suramin
treatment attenuated or abolished tyrosine-phosphorylation of p46, as
well as of p52. Circumstantial evidence that the state of Ras activity
was affected by suramin-induced phenotypic reversion was obtained from
experiments on Raf. In PDGF-stimulated NIH 3T3 cells, as well as
untreated Sis 3T3 cells, Raf-1 was activated as judged from
phosphorylation of recombinant Raf-1 substrate, MEK. Suramin treatment
of Sis 3T3 cells was paralleled by a loss of constitutive Raf-1
activity. These data are in agreement with previous studies showing
that phosphorylation and activation of MEK is necessary and sufficient
for transformation of NIH 3T3 cells
(61) .
-receptor substrates are indeed activated by intracellular
receptors, inaccessible to suramin treatment. However, a likely
explanation of our findings is that signals critical for transformation
are generated at the cell surface. This view is supported by the
finding that the autocrine pathway in V- sis-transformed cells
can be blocked by PDGF antibodies
(64) . We consider this issue
important because of the putative therapeutical implications; autocrine
activation of PDGF receptors is a common trait in human sarcomas and
gliomas and may contribute to their neoplastic phenotype.
Table: Quantification of relative level of
phosphorylation of PDGF receptor and different components in Sis 3T3
cells after suramin treatment, as compared to untreated cells
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.