©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Compartmentalization of Autocrine Signal Transduction Pathways in Sis-transformed NIH 3T3 Cells (*)

Sigrdur Valgeirsdóttir (§) , Anders Eriksson , Monica Nistér (1), Carl-Henrik Heldin , Bengt Westermark (1), Lena Claesson-Welsh

From the (1) Ludwig Institute for Cancer Research, Biomedical Center, Box 595, S-751 24 Uppsala and the Department of Pathology, University Hospital, S-751 85 Uppsala, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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- 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.


INTRODUCTION

PDGF() 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.

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 -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) .

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 -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.


MATERIALS AND METHODS

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 pp60was 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.

I-PDGF Binding Assay

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-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-cmflasks 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 NaVO; 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 NaVO. 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 NaVO, 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 NaVO. 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 NaVO, 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-, GAP, Src, and Shc

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 NaVO. 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 NaVOand 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.


RESULTS

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/10cells) 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.

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 [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-, RasGAP, and Shc

The effect of suramin on tyrosine phosphorylation of PLC-, 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 Caand 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).

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- 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.

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 -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.




DISCUSSION

This investigation is focused on the characteristics of PDGF -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.

Analysis of the state of tyrosine phosphorylation of the PDGF -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) .

Analysis of tyrosine phosphorylation or state of activation of a panel of known PDGF -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) .

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 -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



FOOTNOTES

*
This work was supported in part by grants from the Swedish Institute, the Axel and Margaret Ax:son Johnson Foundation, and the Swedish Cancer Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: PDGF, platelet-derived growth factor; SSV, simian sarcoma virus; mAb, monoclonal antibody; PLC, phospholipase C; GAP, GTPase-activating protein; PI, phosphatidylinositol; PI 3-K, PI 3-kinase; PTP, protein-tyrosine phosphatase; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; PDGFR, PDGF receptor; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; MEK, mitogen-activated protein kinase kinase.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Ross, R., Raines, E. W., and Bowen-Pope, D. F. (1986) Cell 46, 155-169 [Medline] [Order article via Infotrieve]
  2. Claesson-Welsh, L. (1993) in Biology of Platelet-derived Growth Factor: Cytokines (Westermark, B., and Sorg, C., eds) Vol. 5, pp. 31-43, S. Karger AG, Basel
  3. Devare, S. G., Reddy, E. P., Law, J. D., Robbins, K. C., and Aaronson, S. A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 731-735 [Abstract]
  4. Waterfield, M. D., Scrace, G. T., Whittle, N., Stroobant, P., Johnsson, A., Wasteson, Å., Westermark, B., Heldin, C.-H., Huang, J. S., and Deuel, T. F. (1983) Nature 304, 35-39 [Medline] [Order article via Infotrieve]
  5. Doolittle, R. F., Hunkapiller, M. W., Hood, L. E., Devare, S. G., Robbins, K. C., Aaronson, S. A., and Antoniades, H. N. (1983) Science 221, 275-277 [Medline] [Order article via Infotrieve]
  6. Nistér, M., Claesson-Welsh, L., Eriksson, A., Heldin, C.-H., and Westermark, B. (1991) J. Biol. Chem. 266, 16755-16763 [Abstract/Free Full Text]
  7. Hermanson, M., Funa, K., Hartman, M., Claesson-Welsh, L., Heldin, C.-H., Westermark, B., and Nistér, M. (1992) Cancer Res. 52, 3213-3219 [Abstract]
  8. Smits, A., Funa, K., Vassbotn, F. S., Beausang-Linder, M., af Ekenstam, F., Heldin, C.-H., Westermark, B., and Nistér, M. (1992) Am. J. Pathol. 140, 639-648 [Abstract]
  9. Antoniades, H. N., Galanopoulos, T., Neville-Golden, J., and O'Hara, C. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3942-3946 [Abstract]
  10. Shamah, S. M., Stiles, C. D., and Guha, A. (1993) Mol. Cell. Biol. 13, 7203-7212 [Abstract]
  11. Vassbotn, F. S., stman, A., Langeland, N., Holmsen, H., Westermark, B., Heldin, C.-H., and Nistér, M. (1994) J. Cell. Physiol. 158, 381-389 [Medline] [Order article via Infotrieve]
  12. Pawson, T., and Gish, G. D. (1992) Cell 71, 359-362 [Medline] [Order article via Infotrieve]
  13. Mori, S., Rönnstrand, L., Yokote, K., Engström, Å., Courtneidge, S. A., Claesson-Welsh, L., and Heldin, C.-H. (1993) EMBO J. 12, 2257-2264 [Abstract]
  14. Kashishian, A., and Cooper, J. A. (1993) Mol. Biol. Cell 4, 49-57 [Abstract]
  15. Rönnstrand, L., Mori, S., Arvidsson, A.-K., Eriksson, A., Wernstedt, C., Hellman, U., Claesson-Welsh, L., and Heldin, C.-H. (1992) EMBO J. 11, 3911-3919 [Abstract]
  16. Valius, M., Bazenet, C., and Kazlauskas, A. (1993) Mol. Cell. Biol. 13, 133-143 [Abstract]
  17. Kazlauskas, A., Feng, G.-S., Pawson, T., and Valius, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6939-6943 [Abstract]
  18. Fantl, W. J., Escobedo, J. A., Martin, G. A., Turck, C. W., del Rosario, M., McCormick, F., and Williams, L. T. (1992) Cell 69, 413-423 [Medline] [Order article via Infotrieve]
  19. Nishimura, R., Li, W., Kashishian, A., Mondino, A., Zhou, M., Cooper, J., and Schlessinger, J. (1993) Mol. Cell. Biol. 13, 6889-6896 [Abstract]
  20. Kashishian, A., Kazlauskas, A., and Cooper, J. A. (1992) EMBO J. 11, 1373-1382 [Abstract]
  21. Arvidsson, A.-K., Rupp, E., N, E., Downward, J., Rönnstrand, L., Wennström, S., Schlessinger, J., Heldin, C.-H., and Claesson-Welsh, L. (1994) Mol. Cell. Biol. 14, 6715-6726 [Abstract]
  22. Betsholtz, C., Johnsson, A., Heldin, C.-H., and Westermark, B. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6440-6444 [Abstract]
  23. Keating, M. T., and Williams, L. T. (1988) Science 239, 914-916 [Medline] [Order article via Infotrieve]
  24. Fleming, T. P., Matsui, T., Molloy, C. J., Robbins, K. C., and Aaronson, S. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8063-8067 [Abstract]
  25. Robbins, K. C., Antoniades, H. N., Devare, S. G., Hunkapiller, M. W., and Aaronson, S. A. (1983) Nature 305, 605-609 [Medline] [Order article via Infotrieve]
  26. stman, A., Rall, L., Hammacher, A., Wormstead, M. A., Coit, D., Valenzuela, P., Betsholtz, C., Westermark, B., and Heldin, C.-H. (1988) J. Biol. Chem. 263, 16202-16208 [Abstract/Free Full Text]
  27. stman, A., Andersson, M., Betsholtz, C., Westermark, B., and Heldin, C.-H. (1991) Cell Regul. 2, 503-512 [Medline] [Order article via Infotrieve]
  28. Yarden, Y., Escobedo, J. A., Kuang, W.-J., Yang-Feng, T. L., Daniel, T. O., Tremble, P. M., Chen, E. Y., Ando, M. E., Harkins, R. N., Francke, U., Fried, V. A., Ullrich, A., and Williams, L. T. (1986) Nature 323, 226-232 [Medline] [Order article via Infotrieve]
  29. Claesson-Welsh, L., Hammacher, A., Westermark, B., Heldin, C.-H., and Nistér, M. (1989) J. Biol. Chem. 264, 1742-1747 [Abstract/Free Full Text]
  30. Claesson-Welsh, L., Eriksson, A., Morén, A., Severinsson, L., Ek, B., stman, A., Betsholtz, C., and Heldin, C.-H. (1988) Mol. Cell. Biol. 8, 3476-3486 [Medline] [Order article via Infotrieve]
  31. Weima, S. M., van Rooijen, M. A., Mummery, C. L., Feyen, A., de Laat, S. W., and van Zoelen, E. J. J. (1990) Exp. Cell Res. 186, 324-331 [Medline] [Order article via Infotrieve]
  32. Arteaga, C. L., Johnson, M. D., Todderud, G., Coffey, R. J., Carpenter, G., and Page, D. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10435-10439 [Abstract]
  33. Pelicci, G., Lanfrancone, L., Grignani, F., McGlade, J., Cavallo, F., Forni, G., Nicoletti, I., Grignani, F., Pawson, T., and Pelicci, P. G. (1992) Cell 70, 93-104 [Medline] [Order article via Infotrieve]
  34. Lipsich, L. A., Lewis, A. J., and Brugge, J. S. (1983) J. Virol. 48, 352-360 [Medline] [Order article via Infotrieve]
  35. Otsu, M., Hiles, I., Gout, I., Fry, M. J., Ruiz-Larrea, F., Panayotou, G., Thompson, A., Dhand, R., Hsuan, J., Totty, N., Smith, A. D., Morgan, S. J., Courtneidge, S. A., Parker, P. J., and Waterfield, M. D. (1991) Cell 65, 91-104 [Medline] [Order article via Infotrieve]
  36. Bourassa, C., Chapdelaine, A., Roberts, K. D., and Chevalier, S. (1988) Anal. Biochem. 169, 356-362 [Medline] [Order article via Infotrieve]
  37. Cooper, J. A., and Hunter, T. (1981) Mol. Cell. Biol. 1, 165-178 [Medline] [Order article via Infotrieve]
  38. Cooper, J. A., Esch, F. S., Taylor, S. S., and Hunter, T. (1984) J. Biol. Chem. 259, 7835-7841 [Abstract/Free Full Text]
  39. Cook, S. J., and McCormick, F. (1993) Science 262, 1069-1072 [Medline] [Order article via Infotrieve]
  40. Johnsson, A., Betsholtz, C., Heldin, C.-H., and Westermark, B. (1986) EMBO J. 5, 1535-1541 [Abstract]
  41. Claesson-Welsh, L., Rönnstrand, L., and Heldin, C.-H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8796-8800 [Abstract]
  42. Huang, S. S., and Huang, J. S. (1988) J. Biol. Chem. 263, 12608-12618 [Abstract/Free Full Text]
  43. Wennström, S., Landgren, E., Blume-Jensen, P., and Claesson-Welsh, L. (1992) J. Biol. Chem. 267, 13749-13756 [Abstract/Free Full Text]
  44. Kypta, R. M., Goldberg, Y., Ulug, E. T., and Courtneidge, S. A. (1990) Cell 62, 481-492 [Medline] [Order article via Infotrieve]
  45. Berridge, M. J. (1993) Nature 361, 315-325 [CrossRef][Medline] [Order article via Infotrieve]
  46. Valius, M., and Kazlauskas, A. (1993) Cell 73, 321-334 [Medline] [Order article via Infotrieve]
  47. Buday, L., and Downward, J. (1993) Cell 73, 611-620 [Medline] [Order article via Infotrieve]
  48. Egan, S. E., Giddings, B. W., Brooks, M. W., Buday, L., Sizeland, A. M., and Weinberg, R. A. (1993) Nature 363, 45-51 [CrossRef][Medline] [Order article via Infotrieve]
  49. Wennström, S., Siegbahn, A., Yokote, K., Arvidsson, A.-K., Heldin, C.-H., Mori, S., and Claesson-Welsh, L. (1994) Oncogene 9, 651-660 [Medline] [Order article via Infotrieve]
  50. Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991) Cell 64, 281-302 [Medline] [Order article via Infotrieve]
  51. Panayotou, G., and Waterfield, M. D. (1992) Trends Cell Biol. 2, 358-360
  52. Feng, G.-S., Hui, C.-C., and Pawson, T. (1993) Science 259, 1607-1611 [Medline] [Order article via Infotrieve]
  53. Vogel, W., Lammers, R., Huang, J., and Ullrich, A. (1993) Science 259, 1611-1614 [Medline] [Order article via Infotrieve]
  54. Ahmad, S., Banville, D., Zhao, Z., Fischer, E. H., and Shen, S.-H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2197-2201 [Abstract]
  55. Li, W., Nishimura, R., Kashishian, A., Batzer, A. G., Kim, W. J. H., Cooper, J. A., and Schlessinger, J. (1994) Mol. Cell. Biol. 14, 509-517 [Abstract]
  56. Rapp, U. R. (1991) Oncogene 6, 495-500 [Medline] [Order article via Infotrieve]
  57. Crews, C. M., and Erikson, R. L. (1993) Cell 74, 215-217 [Medline] [Order article via Infotrieve]
  58. Marshall, C. J. (1994) Curr. Biol. 4, 82-89 [Medline] [Order article via Infotrieve]
  59. LaRochelle, W. J., Fleming, P., and Aaronson, S. A. (1993) in Biology of Platelet-derived Growth Factor: Cytokines (Westermark, B., and Sorg, C., eds) Vol. 5, pp. 129-145, S. Karger AG, Basel
  60. Satoh, T., Nakafuku, M., and Kaziro, Y. (1992) J. Biol. Chem. 267,24149-24152 [Free Full Text]
  61. Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J. (1994) Cell 77, 841-852 [Medline] [Order article via Infotrieve]
  62. Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700 [Abstract/Free Full Text]
  63. Heldin, C.-H., and Westermark, B. (1990) Cell Regul. 1, 555-566 [Medline] [Order article via Infotrieve]
  64. Johnsson, A., Betsholtz, C., Heldin, C.-H., and Westermark, B. (1985) Nature 317, 438-440 [Medline] [Order article via Infotrieve]

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