Platelet-derived Growth Factor-dependent Cellular Transformation Requires Either Phospholipase Cgamma or Phosphatidylinositol 3 Kinase*

(Received for publication, December 10, 1996, and in revised form, January 30, 1997)

Kris A. DeMali Dagger §, Craig C. Whiteford , Emin T. Ulug and Andrius Kazlauskas Dagger §par

From the Dagger  Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, § University of Colorado Health Sciences Center, Department of Pharmacology, Denver, Colorado 80262, and  Section of Virology and Oncology, Division of Biology, Kansas State University, Manhattan, Kansas 66506

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Although it has been well established that constitutive activation of receptor tyrosine kinases leads to cellular transformation, the signal relay pathways involved have not been systematically investigated. In this study we used a panel of platelet-derived growth factor (PDGF) beta  receptor mutants (beta -PDGFR), which selectively activate various signal relay enzymes to define which signaling pathways are required for PDGF-dependent growth of cells in soft agar. The host cell line for these studies was Ph cells, a 3T3-like cell that expresses normal levels of the beta -PDGFR but no PDGF-alpha receptor (alpha -PDGFR). Hence, this cell system can be used to study signaling of mutant alpha PDGFRs or alpha /beta chimeras. We constructed chimeric receptors containing the alpha PDGFR extracellular domain and the beta PDGFR cytoplasmic domain harboring various phosphorylation site mutations. The mutants were expressed in Ph cells, and their ability to drive PDGF-dependent cellular transformation (growth in soft agar) was assayed. Cells infected with an empty expression vector failed to grow in soft agar, whereas introduction of the chimera with a wild-type beta -PDGFR cytoplasmic domain gave rise to a large number of colonies. In contrast, the N2F5 chimera, in which the binding sites for phospholipase Cgamma (PLC-gamma ), RasGTPase-activating protein, phosphatidylinositol 3 kinase (PI3K), and SHP-2 were eliminated, failed to trigger proliferation. Restoring the binding sites for RasGTPase-activating protein or SHP-2 did not rescue the PDGF-dependent response. In contrast, receptors capable of associating with either PLC-gamma or PI3K relayed a growth signal that was comparable to wild-type receptors in the soft agar growth assay. These findings indicate that the PDGF receptor activates multiple signaling pathways that lead to cellular transformation, and that either PI3K or PLC-gamma are key initiators of such signal relay cascades.


INTRODUCTION

Several lines of evidence implicate PDGF1 and its receptor (PDGFR) as key members in the genesis of certain forms of cancer. First, the B chain of the PDGF ligand is identical to the transforming protein of the v-sis oncogene (1). Second, several studies have shown that co-expression of PDGF and its receptor results in cellular transformation (2), whereas expression of dominant negative constructs of the PDGF reagents can reverse the transformed phenotype of naturally occurring tumor cell lines (3, 4). Third, a fusion between the beta -PDGFR and tel (an ets-like transcription factor) is implicated in the progression of chronic myelogenous leukemia patients to an acute chromic myelomonocytic leukemic state (5). Collectively these findings suggest that deregulation of PDGF-dependent pathways leads to cellular transformation.

There are three isoforms of the PDGF ligand, PDGF AA, PDGF BB, and PDGF AB, which differ in their transforming efficiencies (6, 7). PDGF AA activates only the alpha -PDGFR isoform, whereas the PDGF BB ligand activates both alpha -PDGFRs and beta -PDGFRs (8). The PDGF BB ligand is functionally identical to v-sis (1) and can drive cellular transformation in NIH3T3 cells. In contrast, PDGF AA is less potent at mediating this response. The ability of the PDGF BB ligand to drive cellular transformation of NIH3T3 cells more efficiently than the PDGF AA ligand is believed to be due to either activation of the beta -PDGFR or due to the simultaneous activation of both types of receptors.

The majority of efforts to elucidate beta -PDGFR signal relay has focused on the role of the receptor-associated proteins in mediating regulated growth. In contrast, relatively little is known regarding the signal transduction pathways important for driving abnormal, unregulated growth akin to that which would be present in a cancerous or transformed state. Careful studies comparing the alpha - and beta -PDGFRs suggest that the beta -PDGFR more efficiently transforms cells and that a region in the tail of the beta -PDGFR is critical for this effect (6, 9). It has not yet been determined what signaling pathways are being modulated by this region of the beta -PDGFR to enhance the transformation response.

To better understand the role of PI3K, RasGAP, SHP-2, and PLC-gamma in PDGF-dependent cellular transformation, we constructed a panel of chimeric PDGFR mutants. Each construct contained the extracellular alpha -PDGFR domain and the intracellular beta -PDGFR domain with a tyrosine to phenylalanine substitution at the tyrosine(s) required for binding the receptor-associated proteins. The chimeric constructs were stably expressed in fibroblast cells lacking alpha -PDGFRs and evaluated for their ability to promote PDGF-dependent transformation by assaying growth in soft agar. The chimeric receptor that binds all of the PDGFR-associated proteins, N2WT, was capable of driving PDGF-dependent foci formation, whereas the chimera in which the binding sites for PI3K, RasGAP, SHP-2, and PLC-gamma were mutated failed to promote growth in soft agar. These observations suggested that activation of the kinase activity of the receptor was not enough to drive cellular transformation, and that one or more of the signaling enzymes recruited to the receptor are required. Using the panel of beta -PDGFR mutants, we found that activation of either the PI3K or PLC-gamma signaling cascades restored PDGF-dependent growth in soft agar. We conclude that similar signaling cascades are used to drive normal as well as deregulated growth of cells.


MATERIALS AND METHODS

Cell Lines

The Ph cell line is derived from mouse embryos homozygous for the Ph/Ph deletion that includes the alpha -PDGFR gene (10) and was kindly provided by Dan Bowen-Pope (University of Washington). These are 3T3-like cells that express endogenous beta -PDGFR at approximately 1 × 105 beta -PDGFRs/cell (11) and no alpha -PDGFR. Ph cells were maintained in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 5% calf serum, and 1 mg/ml G418 was added to cultures of cells expressing introduced chimeric constructs. The BALB/C 3T3/v-sis-transformed cells were maintained in DMEM supplemented with 5% calf serum.

Construction of Chimeric PDGFRs

A SacII site was introduced in the human alpha -PDGFR at position 1972 using site-specific mutagenesis according to the protocol supplied with the Amersham Corp. oligonucleotide-directed in vitro mutagenesis system. The mutagenic oligonucleotide had the following sequence: 5'-TAGTCCATCCCGCGGAAACTCCCA-3'. This unique SacII site was just upstream of the kinase domain and was introduced to facilitate the construction of the chimeras with the human beta -PDGFR, which has a naturally occurring SacII site at the analogous position (position 2145). The chimeras were constructed by replacing the entire kinase, kinase insert, and tail regions of the alpha -PDGFR (from the SacII site at position 1972 to the BamHI site at position 3519 of the human alpha -PDGFR) with the corresponding portion of the beta -PDGFR (from the SacII site at position 2145 to the XbaI site at position 4466 of the human beta -PDGFR). In addition to making the wild-type chimera, we also constructed a panel of chimeras in which the intracellular beta -PDGFR domain was one from the series of mutants (F5, Y740/51, Y771, Y1009, or Y1021), the construction of which has been previously described (12). The full-length chimeric constructs were then subcloned into the pLXSN2 retroviral vector, which is a modification of the pLXSN vector (13), such that the polylinker contains the following restriction sites: EcoRI, NotI, HpaI, SalI, and BamHI. The DNA constructs were introduced first into GP+E and then PA317 virus-producing cell lines, or alternatively the DNA was transiently co-transfected with SV-phi--env- and SV-A-MLV-env constructs into the 293T virus-producing cell line (14). The resulting virus was used to infect Ph cells as described previously (12). The receptor-expressing cells were then selected in DMEM, 5% calf serum, and 1 mg/ml G418. The Ph cells expressing the chimeric receptors were subjected to fluorescence-activated cell sorting analysis using an antibody against the extracellular domain of the alpha -PDGFR (PR292) followed by staining with an anti-mouse secondary antibody coupled with fluorescein isothiocyanate fluorescent dye. The cells were sorted so that the chimeric receptors were expressed at approximately the same number as the endogenous receptors, 1 × 105 receptors/cell (10). Periodic assessment of the level of receptor expression by Western blot analysis indicated that the levels of expression were stable for at least several months.

Growth of Cells in Soft Agar

Subconfluent cultures of Ph cells expressing the various receptor constructs were washed gently with phosphate-buffered saline, trypsinized briefly, and resuspended in DME, 5% calf serum, and 0.45% low melting point agarose (SeaPlaque) supplemented with buffer (10 mM acetic acid and 2 mg/ml bovine serum albumin), 200 ng/ml PDGF AA, or 200 ng/ml PDGF BB. These cells were plated into 35-mm tissue culture plates containing a solidified bottom layer of DMEM, 5% calf serum, and 0.6% low melting point agarose. The soft agar cultures were then placed at 37 °C in 5% CO2 for 8-10 days, after which time the foci were counted. Foci larger than eight cells were scored as a colony, and the number of foci in a defined fraction of the dish were counted and used to calculate the number of foci per dish. Three areas of a dish were selected at random for counting the foci, and each condition was assayed in triplicate.

Metabolic Labeling of Cells with myo-[3H]Inositol

To determine cellular levels of inositol metabolites, cells were plated in triplicate at a density of 6 × 105 cells/35-mm well or 1.2 × 106 cells/60-mm tissue culture dish. When nearly confluent, cultures were washed twice with inositol-free DMEM containing 5 µg/ml transferrin and then incubated in this medium for 4-6 h to deplete cellular stores of inositol. Cells were then metabolically labeled by addition of 10-40 µCi of myo-[2-3H]inositol (115 mCi/mmol, Amersham) for 18-24 h. When used for inositol turnover analysis, cultures were treated with LiCl (20 mM) for 30 min prior to addition of growth factor. Cultures were incubated in the presence or absence of human recombinant PDGF AA or BB (40 ng/ml) for 30 min to measure inositol turnover or for 3 min to quantitate D-3-phosphoinositide levels.

Extraction of Inositol Metabolites from Cells

Radioactively labeled cultures were rapidly rinsed with ice-cold phosphate-buffered saline and then treated with 0.6 ml of perchloric acid (4.5%, v/v) at 4 °C. After 15 min, cells were scraped from the dishes with a rubber policeman, transferred to microfuge tubes, and centrifuged at 14,000 rpm for 10 min at 4 °C. Supernatant fluids (containing the inositol phosphates) were transferred to tubes containing 0.12 ml of 10 mM EDTA and 0.5 ml of tri-N-octylamine/freon (1:1, v/v) (15). These tubes were mixed vigorously and centrifuged, and the upper phase (containing inositol phosphates) was transferred to clean tubes and stored at -20 °C.

Perchloric acid-insoluble material (containing phosphoinositides) was washed once with 100 mM EDTA at 4 °C and then resuspended in 50 µl of H2O. Lipids were deacylated by incubating these samples with 1 ml of methanol, 40% methylamine, and n-butanol (4:4:1, v/v) for 45 min at 56 °C (15). After drying, samples were resuspended in 1 ml of H2O, and aliquots were removed to determine the total lipid-associated radioactivity. These glycerophosphoinositide-containing preparations were then extracted twice with butanol/petroleum ether/ethyl formate (20:4:1), dried, and frozen at -20 °C.

HPLC Separation of Glycerophosphoinositides

Glycerophosphoinositide composition of the deacylated lipid preparations was analyzed by anion exchange HPLC using a Whatman Partisphere 5-SAX column. Briefly, glycerophosphoinositide preparations were reconstituted with water, filtered, and then fractionated by HPLC using a series of ammonium phosphate elution gradients designed to isolate isomers within glycerophosphoinositide classes (16). Fractions (0.4 ml) eluting from the column were collected directly into scintillation vials, and 1.2 ml of scintillation mixture (Uniscint BD; National Diagnostics, Manville, NJ) was added to each. Radioactive content of the samples was determined using a Beckman Instruments LS 6000 counter calibrated to correct for quenching and background. Sample recovery using this procedure was greater than 90%. Peaks of interest were identified by co-migration with 32P-labeled lipid standards, prepared as described previously (17).

Analysis of Inositol Turnover

Inositol phosphate-containing preparations were diluted 10-fold with water and applied to columns containing 0.5 ml of anion-exchange resin AG-1, formate form (X8, 200/400 mesh; Bio-Rad). Columns were washed with 30 ml of H2O and 15 ml of 5 mM disodium tetraborate and 60 mM sodium formate to remove free inositol and glycerophosphoinositol, respectively, and then inositol phosphates were eluted with 15 ml of 1 M ammonium formate and 0.1 M formic acid (18). HPLC analysis revealed the presence of inositol phosphate, inositol bisphosphate, and inositol trisphosphate isomers in the inositol phosphate preparations (data not shown). Radioactivity associated with the inositol phosphate fractions was quantitated by liquid scintillation counting and normalized to the total uptake of label into cellular phosphoinositides, to compensate for any differences in inositol uptake or pool size between the different cell lines. Statistical significance was determined by t test using the StatView statistics program (BrainPower, Inc., Calabasas, CA).

Immunoprecipitation and Western Blot Analysis

Subconfluent (85-90%) Ph cells were starved for 18-24 h in DMEM and 0.1% calf serum and then stimulated with 50 ng/ml PDGF AA for 5 min. The cells were then washed and lysed, and the chimeric receptors were immunoprecipitated using a mouse monoclonal antibody directed against the extracellular domain of the human alpha -PDGFR (PR292). The immunoprecipitates were then washed as described previously (19) and subjected to Western blot analysis (12). The blots were developed with Western blot detection reagents (ECL), and the signal was detected by autoradiography.

In Vitro Kinase Assay

Immunoprecipitates were incubated in the presence of 20 mM PIPES, pH 7.0, 10 mM MnCl2, 20 µg/ml aprotinin, and 10 µCi of [gamma -32-P]ATP for 10 min at 30 °C in the presence or absence of 0.5 µg of an exogenous substrate, glutathione S-transferase-PLC-gamma . The fusion protein included amino acid residues 550-850 of rat PLC-gamma . The reaction was stopped by adding an equal volume of 2 × sample buffer (10 mM EDTA, 4% SDS, 5.6 mM 2-mercaptoethanol, 20% glycerol, 200 mM Tris-HCl, pH 6.8, and 1% bromphenol blue). The samples were then incubated for 3 min at 95 °C, spun, and resolved on 7.5% SDS-polyacrylamide gel electrophoresis, and the radiolabeled proteins were detected by autoradiography.


RESULTS

The Chimeric Series of Add-back Mutants

One limitation to studying the role of the PDGFR-associated proteins in driving cellular transformation has been the lack of a suitable cell line. To date, most studies using mutant beta -PDGFRs have been done in cell lines that do not naturally express the PDGFR. The ideal cell line to assay cell transformation would be NIH3T3 cells; however, they have endogenous alpha - and beta -PDGFRs, which limits their suitability for studying introduced beta -PDGFRs. Ph cells, generated from the embryos of the Ph/Ph mouse, are 3T3-like cells that express the beta -PDGFR but not the alpha -PDGFR. Since PDGF AA binds only alpha -PDGFRs, alpha -PDGFRs introduced in Ph cells can be selectively activated with PDGF AA. However, since we were interested in studying the role of the beta -PDGFR-associated proteins in driving PDGF-dependent transformation, we constructed chimeric PDGFRs in which the extracellular, transmembrane, and juxtamembrane regions of the alpha -PDGFR were fused with the intracellular domain of the beta -PDGFR, as shown in Fig. 1A. The panel of chimeras we constructed included the wild-type PDGFR (N2WT), as well as constructs in which the intracellular beta -PDGFR domain harbored the add-back series of beta -PDGFR mutants (Fig. 1B and Ref. 12). This series of mutant receptors was constructed by mutating the intracellular tyrosine residues required for binding PI3K, RasGAP, SHP-2, and PLC-gamma from tyrosine to phenylalanine to generate the F5 receptor, which is unable to associate with any of these proteins. The tyrosine to phenylalanine mutations were then restored one at a time to construct the panel of mutants that associate with one of the signaling molecules (PI3K, RasGAP, SHP-2, or PLC-gamma ) (Fig. 1B).


Fig. 1. Schematic of the alpha /beta chimeric PDGFR constructs. A, the chimeric receptor is constructed such that the extracellular, transmembrane, and juxtamembrane regions are alpha -PDGFR, whereas the remaining portions of the intracellular domain are beta -PDGFR. B, the intracellular domain of the chimeric receptors consists of one of the beta -PDGFR mutants shown here. The speckled shapes represent Src homology 2 domain-containing signal relay enzymes that bind to the phosphorylated receptor, and the filled squares symbolize tyrosine to phenylalanine mutations. Intact phosphorylation sites are represented by P. WT, wild-type beta -PDGFR. N2F5 contains tyrosine to phenylalanine substitutions at tyrosines 740, 751, 771, 1009, and 1021. Each of the names of the members of the mutant series designate which of the phosphorylation sites have been repaired. Y40/51 contains tyrosine at positions 740 and 751 but phenylalanine at the other three phosphorylation sites, whereas Y771 contains tyrosine at position 771 but phenylalanine at the other four phosphorylation sites, etc.
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Although PDGF AA will not activate the endogenous beta -PDGFRs, these receptors could potentially contribute to signaling if the introduced receptors were able to activate the endogenous receptor. To investigate this possibility we examined whether the endogenous beta -PDGFR was activated following stimulation of an introduced alpha -PDGFR. These studies were performed with Ph cells (Phalpha WT) expressing an introduced alpha -PDGFR instead of the chimeric receptor, because we were unable to locate suitable antibodies that distinguish between the chimeric receptor and the endogenous beta -PDGFR. Cultures of Phalpha WT cells (11) expressing an introduced alpha -PDGFR as well as the endogenous beta -PDGFR were either left resting or stimulated with 50 ng/ml PDGF AA for 5 min. The cells were lysed, and the lysates were divided into two equal parts and immunoprecipitated with antibodies specific for either the alpha - or beta -PDGFR, and the immunoprecipitates were analyzed by anti-phosphotyrosine Western blotting. PDGF AA stimulated robust tyrosine phosphorylation of the alpha -PDGFR but no detectable phosphorylation of the beta -PDGFR (Fig. 2, lanes 3-6). To verify that the endogenous beta -PDGFR could be activated, we exposed the Ph parental cell lines to PDGF BB, immunoprecipitated the beta -PDGFR, and subjected it to anti-phosphotyrosine Western blotting. Consistent with previous findings (11), the endogenous beta -PDGFR underwent extensive tyrosine phosphorylation (Fig. 2, lanes 1 and 2). These studies indicate that exposure of Ph cells to PDGF AA does not result in detectable activation of the endogenous beta -PDGFR, suggesting that the beta -PDGFR is not directly activated by PDGF AA or trans-activated by the activated alpha -PDGFR.


Fig. 2. Effect of stimulation of alpha -PDGFRs on activation of beta -PDGFRs. The parental cells (Ph) or Ph cells expressing the wild-type human alpha -PDGFR (Phalpha WT) were grown to 80-90% confluence and starved in DMEM and 0.1% calf serum for 18-24 h (11). The Phalpha WT cells were stimulated with PDGF AA (50 ng/ml) for 5 min, lysed, and divided into two halves that were immunoprecipitated with either an anti-alpha -PDGFR (anti-alpha PR, right panel, lanes 3 and 4) or an anti-beta -PDGFR (anti-beta PR, right panel, lanes 5 and 6) antibody. The Ph cells were stimulated with PDGF BB (40 ng/ml) for 5 min and lysed, and the lysates were immunoprecipitated with the beta -PDGFR-specific antibody (30A, left panel, lanes 1 and 2). The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis followed by anti-phosphotyrosine Western blot analysis.
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The chimeras shown in Fig. 1B were constructed and subcloned into the retroviral expression vector pLXSN2, and recombinant retroviruses were used to infect Ph cells. Mass populations of G418-resistant cells were grown out and sorted by fluorescence-activated cell sorting analysis using a PDGFR antibody (PR292) against the extracellular domain of the chimera. Receptor levels were adjusted to the expression level of the N2WT cell line, which was comparable to the endogenous level of beta -PDGFRs in Ph cells (10). Receptor expression in the resulting cell lines was verified by Western blot analysis of total cell lysates using an antibody (80.8) directed against the extracellular domain of the alpha -PDGFR (Fig. 3A). No chimera was detected in Ph cells expressing an empty vector, whereas all of the other cell lines expressed similar levels of the panel of the chimeric receptors. Fig. 3A, lower panel, is a RasGAP Western blot of the same samples and demonstrates that a similar amount of total cell lysate was present in all lanes.


Fig. 3. Characterization of the chimeric receptors. A, cell lysates representing approximately 4.0 × 104 cells were subjected to alpha -PDGFR Western blot analysis. PhN2 are Ph cells expressing an empty expression vector; the abbreviations for all other receptors are as in Fig. 1, and the N2 prefix indicates a chimeric receptor. Bottom panel, GAP Western blot performed on the same samples, included to normalize for the amount of cell lysate present in the samples. B, Ph cells expressing the chimeric constructs were grown to approximately 85-90% confluence and were left resting (-) or stimulated (+) with 50 ng/ml PDGF AA for 5 min. The Ph cells were washed and lysed, and the chimeric receptors were immunoprecipitated with an antibody against the extracellular alpha  portion of the chimera. Immunoprecipitates, representing approximately 1.5 × 105 cells, were subjected to an in vitro kinase assay in the presence of an exogenous substrate, glutathione S-transferase-PLC-gamma , resolved by SDS-polyacrylamide gel electrophoresis, and subjected to autoradiography. The position of molecular mass standards (in kDa) are shown on the right, whereas the molecular masses of the PDGFR-associated proteins are indicated on the left. The p148, p124, p85, and p64 species are most probably PLC-gamma , rasGAP, p85 subunit of PI3K, and SHP-2, respectively.
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Characterization of the Chimeras

To assess the intrinsic kinase activity of the chimeric constructs, we analyzed their protein kinase activity in receptor immunoprecipitates from resting or stimulated cells. Ph cells expressing the chimeric receptors were grown to 85-90% confluence, starved in DMEM and 0.1% calf serum for 18-24 h, stimulated for 5 min with 50 ng/ml PDGF AA, lysed, and the chimeras were immunoprecipitated using PR292. The immunoprecipitates were subjected to an in vitro kinase assay in the presence of the exogenous substrate, glutathione S-transferase-PLC-gamma , the proteins were resolved by SDS-polyacrylamide gel electrophoresis, and the gel was exposed to film. No detectable receptor kinase activity was recovered from the empty vector-expressing cells, indicating that PR292 did not recognize the endogenous beta -PDGFR (Fig. 3B, lanes 1 and 2). Immunoprecipitates from all of the chimera-expressing cell lines had readily detectable kinase activity, as indicated by phosphorylation of the receptor itself, as well as the exogenous substrate. In addition, the kinase activity of all of the receptor mutants was stimulated by the addition of PDGF prior to cell lysis. The unequal kinase activity of the different receptors was due to variability in the amount of receptor in the samples (data not shown) and was not routinely observed. These findings indicate that creation of a chimeric receptor construct did not impair the intrinsic kinase activity of the receptor.

The autoradiogram in Fig. 3 also addresses the ability of the PDGFR mutants to recruit Src homology 2 domain-containing signal relay enzymes, since the receptor-associated proteins which co-immunoprecipitate with the receptor are phosphorylated in the in vitro kinase assay. After stimulation of cells with PDGF, the N2WT chimera co-immunoprecipitated with p148, p124, p85, and p64 species, which are most probably PLC-gamma , RasGAP, the p85 subunit of PI3K, and SHP-2 (Fig. 3, lanes 3 and 4). In contrast, the N2F5 chimera bound PLC-gamma , p85, and RasGAP poorly (Fig. 3, lanes 13 and 14). Although Tyr1009, which has been previously shown to be required for SHP-2 binding, was mutated in the N2F5 receptor, SHP-2 bound to the N2F5 receptor to near wild-type levels. Restoring the tyrosine residue at position Tyr1009 did not greatly augment binding of SHP-2, and all of the chimeras were able to bind SHP-2 (Fig. 3B). Restoring tyrosines at position 1021 or 771 or both 740 and 751 selectively restored the ability of the PDGFR to associate with PLC-gamma , RasGAP, or PI3K, respectively. Similar results were obtained when the presence of PLC-gamma , RasGAP, PI3K, and SHP-2 in these receptor immunoprecipitates was monitored by Western blot analysis (data not shown). We conclude that with the exception of SHP-2, the binding of the other PDGFR-associated proteins examined can be modulated by mutating receptor phosphorylation sites.

PI-3 Kinase and PLC-gamma Activity

To ensure that the chimeric receptors were able to selectively activate the signaling enzyme with which they associated, PDGF-dependent production of PI3K and PLC-gamma products were examined. To assay production of PI3K products, Ph cells expressing mutant receptors were labeled with myo-[3H]inositol and serum-starved. After 24 h, the cells were stimulated with PDGF AA for 3 min, and the D-3-phosphoinositides were extracted and quantitated as described under "Materials and Methods." In unstimulated cells, there was a comparable basal level of phosphatidylinositol-3,4,5-trisphosphate detected in all of the cell lines examined (Fig. 4A). Exposure to PDGF AA resulted in a marked increase in the level of phosphatidylinositol-3,4,5-trisphosphate and phosphatidylinositol-3,4-bisphosphate in cells expressing the N2WT and N2Y40/51 chimeras, but not the N2F5 or N2Y1021 receptors (Fig. 4A and data not shown). These studies demonstrate that although there is some difference in the relative amounts of products formed, only the receptors that recruit PI3K are able to mediate this event.


Fig. 4. Activation of PI3K and PLC-gamma . Confluent Ph cells were incubated in the presence of inositol-free media to deplete cellular stores of inositol followed by labeling with myo-[3H]inositol for 18-24 h. A, to quantitate D-3-phosphoinositide levels, the cells were left resting (-) or stimulated (+) with PDGF AA (40 ng/ml) for 3 min and treated with perchloric acid. Then the cells were harvested, the lipids were deacylated, and the resulting products were separated by anion exchange HPLC and quantitated using scintillation counting. Phosphatidylinositol-3,4,5-trisphosphate levels in the cell lines are expressed as a fraction of the total cellular phosphoinositides. B, quiescent cultures of Ph cells were pretreated for 30 min with LiCl and then left resting (-) or stimulated (+) with PDGF AA (40 ng/ml). Inositol phosphates were extracted from cells with perchloric acid and purified by anion-exchange chromatography. Inositol phosphates were quantitated by scintillation counting and expressed as a fraction of the total cellular phosphoinositides.
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We next examined PDGF-dependent PLC-gamma activation by measuring the accumulation of PLC-gamma products in intact cells. Cells were incubated with myo-[3H]inositol for 18-24 h and then stimulated with PDGF AA for 30 min in the presence of LiCl. Inositol phosphates were extracted and purified by anion-exchange chromatography. PDGF stimulated a robust increase in inositol turnover in the N2WT and the N2Y1021 chimera-expressing cells (Fig. 4B). In contrast, the N2F5 and N2Y40/51 receptors were unable to activate PLC-gamma . Note that the level of inositol phosphates produced in response to stimulation of the N2Y1021 chimera was consistently greater than that observed on stimulation of the N2WT chimera. A possible explanation is that some of the other proteins that associate with the N2WT receptor but not the N2Y1021 receptor negatively regulate PLC-gamma activation (20). Taken together these studies indicate that the chimeric receptor mutants that bind PI3K or PLC-gamma selectively activate the PI3K or PLC-gamma signaling pathways, respectively.

Role of the Receptor-associated Proteins in Driving Cellular Transformation

To examine the contribution of the various signaling pathways in PDGF-dependent cellular transformation, we determined the ability of the receptor mutants to promote growth of cells in soft agar. Cells were plated in soft agar containing buffer alone, PDGF AA, or PDGF BB. PDGF AA was used to measure foci formation in response to activation of the introduced chimeric receptors, whereas PDGF BB activates both the endogenous and introduced PDGF receptors, so the resulting PDGF-dependent soft agar growth reflects the contribution of all PDGFRs. Foci were photographed and quantitated after 8-10 days of incubation at 37 °C and 5% CO2.

Cells expressing the empty vector did not grow in agar containing buffer or PDGF AA, whereas an average of 3113 colonies were detected in a 35-mm plate of cells supplemented with 200 ng/ml PDGF BB (Fig. 5). Like the empty vector-expressing cells, the cells harboring the N2WT chimeras failed to grow into detectable colonies when plated in the absence of PDGF (Fig. 5). Unlike the control cells, however, PDGF AA stimulated a robust response in these WT receptor-expressing cells, (5894 colonies/35-mm dish). Thus like the endogenous beta -PDGFRs, the N2WT chimeric PDGFR was able to trigger growth in soft agar. In contrast, no colonies were observed when cells expressing the N2F5 receptor were cultured in the presence of PDGF AA (Fig. 5), indicating that the proteins that still bind to N2F5 are not sufficient to drive PDGF-dependent transformation.


Fig. 5. Comparison of the PDGF-stimulated growth of Ph cells expressing the chimeric receptors. Ph cells infected with an empty vector (PhN2), the wild-type chimera (PhN2WT), or the F5 mutant (PhN2F5) were plated in DMEM and 5% calf serum in the presence of buffer or 200 ng/ml PDGF AA or PDGF BB as denoted at the top. The numbers at the bottom represent the average numbers of colonies from three independent dishes of cells.
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In response to activation using PDGF BB, Ph cells expressing an N2 or N2F5 receptor were able to drive growth in soft agar to similar levels (3113 and 3487 colonies in the N2 and N2F5 dishes, respectively). Also, the morphology of the colonies in both instances was similar. In contrast, the N2WT-expressing cells grow better in response to PDGF BB than either the N2- or N2F5-expressing cells, with approximately 6990 foci observed. The enhanced colony number is likely to be due to the contribution of the chimeric receptor. The morphology of the N2WT colonies was similar to that observed with the N2- and N2F5-expressing cells. Although these studies were done using 200 ng/ml PDGF, smaller doses (100 or 50 ng/ml PDGF AA or PDGF BB) yielded similar results (data not shown).

To compare the response of Ph cells with those of other cell types that have been used by other groups, we analyzed PDGF-dependent soft agar growth of NIH3T3 cells and BALB/C 3T3/v-sis cells. In the absence of PDGF, the NIH3T3 cells did not grow in soft agar, whereas PDGF BB and PDGF AA induced 4162 and 2773 colonies, respectively (Fig. 6). These findings are consistent with reports from other groups showing that PDGF BB is more potent than PDGF AA (6). Unlike the NIH3T3 and Ph cells, BALB/C 3T3/v-sis cells grew well in soft agar without exogenously applied PDGF (Fig. 6). Addition of PDGF to the culture media led to a modest increase in the number of colonies that grew, but the exogenously applied PDGF did not alter size or morphology of the colonies. In comparison, the PhN2WT cells grew better in response to PDGF than the NIH3T3 cells but produced fewer colonies than the BALB/C 3T3/v-sis cells. The morphology of the colonies in all three cell types was similar, except that PhN2WT cells formed larger colonies (Fig. 6). These studies indicated that Ph cells grow in soft agar in response to PDGF stimulation to form colonies with a frequency, size, and morphology comparable to those observed in other cell types. Furthermore, one or more of the signaling pathways engaged by the N2WT but not the N2F5 receptor is required for the soft agar growth response.


Fig. 6. Soft agar growth of PhN2WT, NIH3T3, and BALB/C 3T3/v-sis cells. Cells were plated in DMEM and 5% calf serum in the presence of buffer or 200 ng/ml of PDGF AA or PDGF BB as denoted at the top. The numbers at the bottom represent the average numbers of colonies from three independent dishes of cells.
[View Larger Version of this Image (67K GIF file)]


We next examined whether restoring the binding sites for RasGAP or SHP-2 rescued the ability of the N2F5 chimera to promote growth in soft agar. Activation of chimeric receptors with intact RasGAP or SHP-2 binding sites did not rescue the ability of the receptor to form foci in soft agar (data not shown). Furthermore, in both cell lines, PDGF BB promoted foci formation to a level comparable to the N2F5-expressing cells.

Contrary to the results with N2Y771 and N2Y1009 receptors, restoring the binding sites for either PI3K or PLC-gamma rescued PDGF-dependent growth in soft agar. In the presence of PDGF AA, both the N2Y1021 and N2Y40/51 receptor constructs produced an increase in foci formation over that of buffer alone, with approximately 5494 and 5277 of the cells forming foci, respectively (Fig. 7). The numbers of foci formed were comparable to those observed with the N2WT chimera, suggesting that restoring the binding sites for either PLC-gamma or PI3K can fully restore the ability to grow in soft agar to near wild-type levels. Colonies of N2WT and N2Y1021 cells were morphologically indistinguishable, whereas colonies of N2Y40/51 cells were consistently larger than those produced by the N2Y1021- or N2WT-expressing cells (Fig. 7). Collectively these findings suggest that PI3K or PLC-gamma is required for PDGF-dependent growth in soft agar.


Fig. 7. Result of restoring the binding sites for PLC-gamma or PI3K to the F5 mutant background. Ph cells expressing the wild-type chimera (PhN2WT), a chimera able to bind PLC-gamma (N2Y1021), or a chimera able to bind PI3K (N2Y40/51) were tested for growth in soft agar as described in the legend of Fig. 5.
[View Larger Version of this Image (63K GIF file)]



DISCUSSION

In this study we have evaluated the importance of RasGAP, SHP-2, PLC-gamma , and PI3K in promoting PDGF-dependent growth of mouse Ph fibroblasts in soft agar. We observed that receptors capable of associating with PI3K and PLC-gamma , but not RasGAP or SHP-2, initiate pathways that result in foci formation.

There is considerable controversy concerning the role of PI3K and PLC-gamma in PDGF-dependent cell cycle progression. One important variable is that, depending on the type of PDGF used, one or more isoform of the PDGF receptor can be activated. Different signaling pathways are used by the alpha - and beta -PDGFRs; hence, the downstream effects are not identical. Studies by Yu et al. (21, 22) suggest that stimulation of chimeric receptor constructs that contained colony-stimulating factor-1 receptor extracellularly and alpha -PDGFR receptor intracellularly promoted growth in soft agar in response to colony-stimulating factor stimulation. Mutation of one or both of the sites required for binding of PI3K and PLC-gamma to the alpha -PDGFR did not compromise the ability of these chimeric constructs to promote growth in soft agar (21, 22). These findings suggest that for the alpha -PDGFR, PI3K and PLC-gamma are dispensable for PDGF-dependent growth in soft agar. In contrast, using the beta -PDGFR chimera system described here, we find that the beta -PDGFR initiates multiple pathways that lead to growth in soft agar, and that PI3K and PLC-gamma are required to engage these events. The extent to which these pathways converge downstream of PI3K and PLC-gamma remains to be investigated.

In comparison with the alpha -PDGFR, the beta -PDGFR more efficiently stimulates growth of cells in soft agar, and recent studies (9) identified a region in the beta -PDGFR tail that is important for conferring the enhanced transforming activity of the beta -PDGFR. These studies were carried out by swapping a piece of the beta -PDGFR into alpha -PDGFR, which included the PLC-gamma binding site for both of the receptors. As a result the swapped receptor is an alpha -PDGFR with a beta -PDGFR PLC-gamma binding site. The alpha -PDGFRs and beta -PDGFRs have been compared with respect to PLC-gamma binding and activation (23), and based on these findings, one would predict that replacing the PLC-gamma binding site of the alpha -PDGFR with the PLC-gamma binding site of the beta -PDGFR would decrease binding of PLC-gamma and increase tyrosine phosphorylation and activation of PLC-gamma . Although the ability of the swapped receptor to activate PLC-gamma has not been reported, the hybrid receptor should activate PLC-gamma better than wild-type alpha -PDGFR. This is consistent with our observation that PLC-gamma is required for soft agar growth by the beta -PDGFR. However, it is not consistent with findings by Obermeier et al. (24), which suggest that PLC-gamma activation inversely correlates with transformation. Although all of these studies indicate that PLC-gamma is important in cellular transformation, the exact role of this signaling molecule, as well as whether it has a positive or negative effect, has not yet emerged.

An unexpected observation that arose from our studies was the ability of the chimeric receptors to bind SHP-2 independent of phosphorylation of tyrosine 1009 (12, 25). Previous studies have shown that mutating the tyrosine at position 1009 in the WT receptor largely eliminates SHP-2 binding, whereas restoring tyrosine at 1009 in the F5 receptor enables the phosphorylated beta -PDGFR to bind SHP-2 stably (12). One difference between these studies is the host cell line in which these receptor constructs were expressed. The chimeric receptors were expressed in Ph cells, whereas the beta -PDGFR mutants were expressed in HepG2 cells. Consequently, we tested whether the unusual binding of SHP-2 was cell line-dependent and found that this was not the case.2 Although SHP-2 binding is still dependent on tyrosine phosphorylation of the receptor, it appears that the chimera is able to bind SHP-2 in a Tyr1009-independent manner. It is possible that creation of the chimeric receptors alters receptor conformation such that some sequences that were previously unavailable for SHP-2 binding are now accessible. Studies are currently under way to understand better the binding of SHP-2 to the chimeric PDGFRs independent of an intact binding site.

Although SHP-2 associated with all of the receptors used in these studies, we do not think that this changes the interpretation of the soft agar data. Previous studies from our laboratory have suggested that SHP-2 does not play a central role in relaying signals that lead to a proliferative response (20, 26). Furthermore, restoring the binding site for SHP-2 to beta -PDGFRs capable of associating with PLC-gamma had no effect on PLC-gamma binding, activation of PLC-gamma , or the DNA synthesis response (20). Similarly, restoration of the SHP-2 binding site to receptors capable of associating with PI3K did not inhibit PDGF-dependent activation of PI3K (27). Thus SHP-2 binding does not negatively affect signaling by either PLC-gamma or PI3K, and binding of SHP-2 itself does not mediate DNA synthesis. These data are consistent with the idea that SHP-2 may not play a pivitol role in regulating the PDGF-dependent growth of cells in soft agar.

Although we have focused on binding of PI3K, PLC-gamma , SHP-2, and RasGAP to the chimeric receptor mutants described in these experiments, it is likely that several other proteins associate with these receptors as well. For instance, Src associates with the F5 receptor to near-WT levels when expressed in HepG2 or A431 cell lines.3 The chimeric N2F5 receptor was unable to mediate PDGF-dependent growth of cells in soft agar, indicating that Src and other proteins that may associate with the N2F5 receptor are not sufficient to trigger a biological response. However, it is possible that Src or these other receptor-associated proteins play a co-operative role in mediating this response.

In summary, these studies suggest that PI3K or PLC-gamma binding to chimeric alpha /beta receptors is required for PDGF-dependent growth of cells in soft agar. Previous reports (12) have suggested that activation of PI3K or PLC-gamma is sufficient to promote PDGF-dependent DNA synthesis. The data present herein indicate that PI3K or PLC-gamma are required for PDGF-dependent transformation and suggest that the early steps in the pathways leading to normal or cancerous growth are surprisingly similar.


FOOTNOTES

*   This research was supported in part by National Institutes of Health Grants CA58291 (to E. T. U.) and GM-48339 (to A. K.) and by a grant-in aid from the American Heart Association, Kansas Affiliate (to E. T. U.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    An Established Investigator of the American Heart Association. To whom correspondence should be addressed: Schepens Eye Research Institute, Harvard Medical School, 20 Staniford St, Boston, MA 02114. Tel.: 617-723-6078 (ext. 517); Fax: 617-523-3463; E-mail, kazlauskas{at}vision.eri.harvard.edu.
1   The abbreviations used are: PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PI3K, phosphatidylinositol 3 kinase; PLC-gamma , phospholipase Cgamma ; DMEM, Dulbecco's modified Eagle's medium; HPLC, high performance liquid chromatography; PIPES, 1,4-piperazinediethanesulfonic acid; WT, wild-type; GAP, GTPase-activating protein.
2   Unpublished observations.
3   J. P. Secrist, J. A. Gelderloos, R. R. Vaillancourt, and A. Kazlauskas, manuscript in preparation.

ACKNOWLEDGEMENTS

We thank Charlie Hart for the PDGF AA, Dan Bowen-Pope for the Ph cells, Chuck Stiles for the BALB/C 3T3/v-sis cells, and members of the Kazlauskas laboratory for critically reviewing the manuscript.


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