EJ-Ras Inhibits Phospholipase Cγ1 but Not Actin Polymerization Induced by Platelet-Derived Growth Factor-BB via Phosphatidylinositol 3-Kinase
Abstract Transformation of fibroblast-like cells (NIH 3T3) by a constitutively activated GTP-bound isoform of p21ras (EJ-Ras) produces morphogenic changes characterized by decreased attachment to the substratum, with retraction and rounding of the cell body. Transformed fibroblasts lose their “stressed” conformation and adopt a “relaxed” morphology. The specific molecular mechanisms responsible for these changes remain uncharacterized. We found that EJ-Ras transformation of NIH 3T3 cells decreased the cellular content of polymerized actin, particularly at the expense of actin stress fibers, but induced the accumulation of actin filaments in peripheral ruffling membranes. Polymerization of actin could be induced in EJ-Ras–transformed cells by exposure to platelet-derived growth factor (PDGF)-BB to an extent similar to that observed in wild-type NIH 3T3 cells. In EJ-Ras cells, actin polymerization was independent of phospholipase Cγ1 (PLCγ1) activity, because inositol tris-phosphate (IP3) production observed in control NIH 3T3 cells in response to PDGF-BB was absent. Although PDGF-BB did stimulate tyrosine phosphorylation of PLCγ1, the phospholipase was strongly inhibited by an inhibitory factor present in the cytoplasm of EJ-Ras–transformed cells. In addition, cytoplasmic extracts of EJ-Ras, but not of control cells, inhibited phosphatidylinositol 4,5-diphosphate (PIP2) hydrolysis catalyzed by a recombinant PLCγ1 in vitro. Although PIP2 hydrolysis could not contribute to the reorganization of the actin cytoskeleton induced by PDGF-BB in EJ-Ras–transformed cells, phosphatidylinositol 3-kinase (PI3-K) was necessary for actin polymerization. Wortmannin, a specific PI3-K inhibitor, not only blocked actin polymerization in both control and EJ-Ras–transformed cells but actually led to rapid actin depolymerization when these cells were exposed to PDGF-BB. Thus, in EJ-Ras–transformed cells, cell morphogenic changes in response to PDGF-BB rely importantly on PI3-K and can occur in the complete absence of IP3 production despite tyrosine phosphorylation of PLCγ1.
Migration of vascular cells from the media to the intima corresponds with an important step in the pathological processes of restenosis and atherogenesis.1 This migration is strongly influenced by PDGF, a potent chemotactic agent for smooth muscle cells and fibroblasts,2 3 and requires a rapid and reversible reorganization of the actin cytoskeleton.4 5 The characterization of intracellular signaling events regulating this coordinated membrane and cytoskeletal response remains incomplete, but in smooth muscle cells, PLCγ1 seems to play a central role.6 Site-directed mutagenesis analysis of PDGF-R has demonstrated that the respective bindings of PI3-K and PLCγ1 to phosphotyrosine residues 708 and 719 and residues 977 and 989 of PDGF-R are both important in the motile response stimulated by PDGF.7 The effect of these initial activation events is also modulated by the activity of small GTP-binding proteins of the p21ras family (eg, Rho, Rac, and Cdc 42).8 9 10 Rac, in particular, is necessary for stabilization of specific actin structures required for membrane ruffling and motility induced by growth factors.9
How small GTP-binding proteins and inositol phospholipids interact in the regulation of the actin cytoskeleton is now better understood.11 12 13 Binding of the fibronectin receptor to its extracellular ligand triggers the activity of phosphatidylinositol 4-phosphate 5-kinase in a GTP-Rho–dependent manner.11 12 Rac association with GTP (the active conformation) is regulated by PI3-K.13 To help characterize further the relationships between these messenger systems, we studied the actin cytoskeleton of NIH 3T3 cells transformed with EJ-Ras, an oncogenic mutant of the cellular Ha-Ras, which produces a constitutive “p21ras-on” state of the transformed cells.14 Previous work has shown that EJ-Ras prevented PDGF signaling by inhibiting tyrosine phosphorylation (or by increasing dephosphorylation) of PDGF-R.15 16 However, new evidence suggested that reduced tyrosine phosphorylation of the PDGF-R could be secondary (because of the relaxed state of cell adhesion) instead of causative in the altered signaling system of EJ-Ras–transformed cells.17 Therefore, we decided to characterize this signaling system further and to study the consequences of signaling alterations in the ability of PDGF-BB to induce an actin response.
We found that EJ-Ras transformation did not completely prevent tyrosine phosphorylation of PDGF-R and had little effect on PLCγ1 tyrosine phosphorylation in response to PDGF-BB. Instead, EJ-Ras transformation produced a soluble factor that completely inhibited PLCγ1 activity and was responsible for inhibiting PIP2 hydrolysis and downstream signaling events. In spite of the absence of stimulated PIP2 hydrolysis in EJ-Ras–transformed cells, PDGF-BB was able to produce actin filament polymerization that was inhibitable by wortmannin, a specific PI3-K inhibitor. In fact, inhibition of PI3-K in both control and EJ-Ras–transformed cells led to depolymerization of actin filaments upon exposure to PDGF-BB. This paradoxical effect strongly suggests that PDGF normally induces concurrent polymerization and depolymerization of actin, with resulting net polymerization. Wortmannin also prevented the increased baseline motility of EJ-Ras–transformed cells and the motile response of these cells to PDGF-BB (L.E.C., A.W.H., P.J.G.-C., R.W.T., unpublished data). Thus, our data support the prime importance of the PI3-K pathway in EJ-Ras–transformed cells in producing morphogenic changes transiently induced by PDGF-BB and constitutively induced by EJ-Ras. These results emphasize the interaction between small GTP-binding proteins and specific enzymes of the inositol signaling pathway and suggest new strategies for the prevention of restenosis and atherogenesis.1
Materials and Methods
DMEM (high glucose), CO2-independent medium, Dulbecco’s PBS, penicillin-streptomycin, FCS, G418 sulfate, trypsin-EDTA, and Lipofectin reagent were from GIBCO. Agarose-conjugated anti-p21ras IgG, and monoclonal anti–Ha-Ras antibodies were from Santa Cruz Biotechnology. Recombinant human PDGF-BB, anti–PDGF-R antibody (type A/B), agarose-conjugated and soluble anti-phosphotyrosine IgG, anti-p85 of PI3-K, and anti-PLCγ1 monoclonal IgG were from Upstate Biotechnology Inc. C4 monoclonal anti-actin antibody was from ICN. Protein A–Sepharose was from Pharmacia. Rhodamine phalloidin was from Molecular Probes. Wortmannin, BSA (radioimmunoassay grade), antipain, pepstatin, chymostatin, and leupeptin were from Sigma Chemical Co. AEBSF was from Calbiochem. Precoated thin-layer chromatography silica H plates with 1% potassium oxalate were from Analtech. Glacial acetic acid was from J.T. Baker, Inc. [3H]PIP2 and [3H]inositol were from Amersham. En3Hance spray (surface autoradiography enhancer) was from Dupont (Biotechnology Systems, NEN Research Products). All chemicals were first grade and, unless otherwise indicated, were from Sigma. Recombinant PLCγ1 was prepared and characterized as previously described.18 Polyacrylamide 4% to 20% gradient gels were from Jule. Horseradish peroxidase–labeled IgGs were from HyClone. The ECL technique (Amersham) was used to develop Western blots. Densitometry was used for the analysis chemiluminograms where quantitative data on the intensity of bands on Western blots are provided.
Constructs and Cell Lines
To obtain EJ-Ras–transformed cell lines, NIH 3T3 cells (American Type Culture Collection) were cotransfected with 20 μg of pUC19/EJ-Ras and 1 μg of pCMVNeo using Lipofectin reagent and the manufacturer’s instructions. G418-resistant control cell lines were obtained by Lipofectin transfection of NIH 3T3 cells with 1 μg of pCMVNeo. The plasmid pUC1-9/EJ-Ras contains a 6.6-kB BamHI fragment with EJ-Ras cDNA and its promoter from the EJ bladder carcinoma cell line.14 The pCMVNeo plasmid encodes resistance to G418.19 Selection of clones was carried out by dilution in 96-well plates in G418 sulfate at 600 mg/L and reduced to 250 mg/L for maintenance of lines. The cell lines 5A2 and 5A6 are representative clonal cell lines expressing EJ-Ras, whereas the EJ-Ras pool corresponds to a mixture of ≈25 G418-resistant clones after transfection with pUC19/EJ-Ras and pCMVNeo. The control cells (Neos) were expanded from ≈20 G418 clones after transfection with pCMVNeo.
Cells were grown in DMEM, to which was added 1% penicillin-streptomycin, 5% FCS, and 250 mg/L G418 sulfate (complete medium). Cells were grown at 37°C in a water-jacketed incubator with a humid 5% CO2 atmosphere. Medium was exchanged twice weekly, and cells were passaged at subconfluence with 0.05% trypsin and 0.53 mmol/L EDTA. Before stimulation with PDGF-BB, cells were maintained for 48 hours in DMEM supplemented with 0.5% FCS (serum-starvation conditions).
The preparation of cell extract was performed according to our standard procedure20 ; briefly, cells were lysed in melting ice-cold buffer containing 145 mmol/L NaCl, 0.1 mmol/L MgCl2, 15 mmol/L HEPES (pH 7.0), 5 mmol/L EGTA, 1 mmol/L AEBSF, 20 mg/L each of chymostatin, leupeptin, antipain, and pepstatin, 1.0 mmol/L sodium vanadate, and 0.5% Triton X-100 (lysis buffer). Cell lysates of 5A2 and 5A6 clones, of the EJ-Ras pool, and of neomycin-resistant controls were normalized for total protein at ≈2 g/L. Cell lysates (2 mL) were incubated with 15 μL of agarose bead–conjugated anti–pan-p21ras IgG for 12 hours at 4°C. Beads were washed with ice-cold PBS and boiled in SDS buffer,20 and the supernatants were electrophoresed on SDS-polyacrylamide gels and then transferred onto nitrocellulose.21 Western blots were incubated with 1:40 mouse monoclonal anti–pan-p21ras IgG or 1:50 rat monoclonal anti–Ha-Ras IgG and then with 1:400 peroxidase-labeled goat anti-mouse or goat anti-rat, respectively; we used the ECL detection system for all Western blots.20
Filamentous Actin Assay
Confluent cells were treated with 30 μg/L PDGF-BB for 1, 3, 9, or 27 minutes or with vehicle buffer (120 mmol/L NaCl, 4 mmol/L KCl, 1 mmol/L KH2PO4, 1 mmol/L MgSO4, 1.3 mmol/L CaCl2, and 1.2 mmol/L HEPES [Krebs/HEPES]+0.1% BSA) for 27 minutes, followed immediately by fixation with 3.7% formaldehyde in PBS for 30 minutes. Fixed cells were permeabilized using 0.2% Triton X-100, and F-actin was stained with 0.2 μmol/L rhodamine phalloidin, a concentration shown to be saturating.20 Unbound fluorescence was washed away, and bound fluorescent indicator was extracted in 0.1 mol/L NaOH overnight. Fluorescence corresponding to the rhodamine phalloidin that was bound to actin filaments was measured at 580-nm (slit, 5 nm) emission and 550-nm (slit, 2 nm) excitation, then was normalized to the total protein content measured by Bradford assay21 in identically treated cells, and was expressed as a fraction of the fluorescent signal measured in unstimulated control cells.
F-actin was also measured using a biochemical assay on microsamples.20 Briefly, 5×105 cells were extracted in ice-cold lysis buffer (150 μL) supplemented with rhodamine phalloidin (0.6 μmol/L, shown to be saturating).20 The extracts were homogenized and normalized and then centrifuged at 150 000g for 60 minutes. Pelleted actin filaments were rinsed once with plain lysis buffer (200 μL). Rhodamine phalloidin was solubilized in methanol (200 μL) overnight at 4°C and then transferred into 96-well microtiter plates (Corning, flat bottom, low protein binding). Fluorescence was measured in a CytoFluor 2300 (Millipore), using excitation at 530 nm and emission at 590 nm.
Total actin was determined for cell lysates by reacting Western blots of normalized extracts with 1:1000 C4 monoclonal anti-actin antibody and 1:500 peroxidase-labeled goat anti-mouse. The blots were developed with the ECL method. The densities of actin bands were compared with those of actin standards to calculate cellular actin concentration.
Fluorescence Microscopy and Confocal Analysis of the Actin Cytoskeleton
Cells cultured on coverslips were treated with 30 μg/L PDGF-BB for 1 or 5 minutes, and the F-actin cytoskeleton was stained with 0.2 μmol/L rhodamine phalloidin. Photomicrography was performed using a Zeiss Axiophot microscope and Ektachrome Elite (ASA 400). Laser scanning confocal microscopy was performed with a BioRad MRC 600, and rhodamine fluorescence was detected with a BioRad GHS filter block. Sequential images (z series) represented slices ≈1.5 μm thick and 1 μm apart in the z axis (into/out of the plane of the slide). Images were stored on optical disk, and artificial color hard copies were generated on a color video printer.
PDGF-R Tyrosine Phosphorylation Assay
PDGF-BB was dissolved (10 mg/L) in aqueous 0.1 mol/L acetic acid and 0.1% BSA and was used at a final concentration of 30 μg/L in PBS to stimulate confluent cells. Serum-starved cells were treated at 37°C with PDGF-BB for 1 to 20 minutes or were treated with vehicle alone for 20 minutes. Stimulation was stopped by scraping cells into melting ice-cold lysis buffer.20 Lysates were sonicated for 10 seconds (at energy level 1 to 2; Sonifier 450, Branson, Inc) and centrifuged at 14 000 rpm for 3 minutes (Eppendorf centrifuge 5415C). Supernatants were normalized for total protein with lysis buffer and incubated with rabbit polyclonal anti-human PDGF-R type A/B antibody for 10 minutes at 4°C and then with protein A–Sepharose for 12 hours. Electrophoresis and Western blotting were performed as described above,21 and blots were reacted with 1:200 anti–PDGF-R antibody and 1:1000 peroxidase-labeled goat anti-rabbit IgG for quantification of total PDGF-R or with 1:1000 mouse monoclonal anti-phosphotyrosine and 1:1000 peroxidase-labeled goat anti-mouse to detect the fraction of tyrosine-phosphorylated receptor.
PLCγ1 Phosphorylation and Enzymatic Assay
Extracts of PDGF-stimulated and of control cells (conditions as above) were immunoprecipitated with agarose-conjugated mouse monoclonal anti-phosphotyrosine (for tyrosine-phosphorylated PLCγ1, 5 μL of beads for 500 μL of extract) or with multimonoclonal antibodies for PLCγ1 and protein A–Sepharose beads (for total PLCγ1, 2 μg of anti-PLCγ1 and 10 μL of beads); the precipitated immune complexes were electrophoresed and blotted, and blots were reacted with 1:1000 multimonoclonal anti-PLCγ1 and 1:1000 peroxidase-labeled goat anti-mouse antibody.
To test for the presence of an inhibitor of PLCγ1 in the cytosol of control and EJ-Ras–transformed cells, cell extracts were added to purified recombinant PLCγ1 (0.14 nmol/L), and total resulting PLC activity (intrinsic to the extract plus exogenous recombinant PLCγ1) was measured on SSLVs.22 23 PLC activity was measured in the presence of cellular extracts obtained from control and EJ-Ras–transformed cells using a lysis buffer containing lower Triton X-100 concentration (0.05%) and, as the substrate, SSLVs containing PIP2 (50 μmol/L, with traces of [3H]PIP2) and phosphatidylethanolamine (1:10 ratio). Each sample contained 5 μL of SSLVs, various volumes of cell extract (as indicated, 1.0 g/L), and 40 μL of vesicle buffer containing 5 mmol/L Tris (pH 7.0), 0.4 mmol/L EGTA, and 0.2 mmol/L CaCl2.22 23 Released [3H]IP3 was isolated by lipid extraction at various time points and quantified in a scintillation counter.22 23
IP3 Competitive Binding Assay
IP3 mass measurements were made using IP3-binding protein prepared from rat cerebellum. Each rat cerebellum was added to 10 mL of Tris-EDTA buffer (50 mmol/L Tris-HCl [pH 7.7], 1 mmol/L EDTA, and 1 mmol/L β-mercaptoethanol). After centrifugation at 13 000 rpm (SS-34 rotor) for 15 minutes, membrane preparations were obtained after washing the pellets twice with Tris-EDTA buffer, followed by resuspension in the same buffer. A standard curve was obtained with known concentrations of [3H]IP3 in Tris-EDTA buffer (adjusted to pH 8.4), such that a linear relationship from 1 to 100 nmol/L IP3 was obtained. Each binding reaction consisted of adding 50 μL of IP3-binding protein preparation to 50 μL of IP3 standards and 400 μL of Tris-EDTA buffer (pH 8.4) in Eppendorf tubes. The reactions were vortexed vigorously for 1 second, incubated for 20 minutes at 0°C to 4°C, and centrifuged for 5 minutes at 14 000 rpm in Eppendorf tubes; the supernatants were then discarded. The pellets were resuspended in 0.5 mL of Tris-EDTA buffer (pH 8.4), washed twice in the same buffer, and then added to scintillation fluid to be counted.
Measurements of IP3 at various time points of cultured cell assays (performed in 35-mm cuvettes) were performed by stopping the reactions with 1 mL of ice-cold 10% trichloroacetic acid, scraping, and sonicating for 10 seconds. The suspensions were then placed on ice and centrifuged for 15 minutes (in Eppendorf tubes, 14 000 rpm). Supernatants were assayed for IP3 content. For the competitive assays, unlabeled IP3 produced by NIH 3T3 cells (both control and EJ-Ras–transformed cells, either quiescent or activated) was mixed with [3H]IP3 (100 nmol/L) in 50-μL samples before mixing with the aliquot of IP3-binding protein. A decrease in 3H count in the pellets indicated that more cold IP3 competed with labeled IP3 for the binding protein.
Inositol Phospholipid Mass Measurements
The mass of major inositol phospholipids in NIH 3T3 cells (PI, PIP, and PIP2) was determined by thin-layer chromatography of extracted lipids. The inositol phospholipids of confluent NIH 3T3 cells in six-well plates were labeled by starving the cells from inositol for 48 hours (inositol-free DMEM, 5% dialyzed FBS) and then adding [3H]inositol to the cultures (10 to 50 μCi). At various time points, the cells were extracted by scraping the cells in methanol/chloroform (2:1 [vol/vol]). The organic extracts were dried under nitrogen, resuspended in 750 μL of methanol/chloroform (2:1), vortexed, added to 500 μL chloroform/1 mol/L HCl (1:1 [vol/vol]), and centrifuged for 5 minutes at 3000 rpm in a Beckman tabletop centrifuge. The lower phase was collected (containing inositol phospholipids), dried under nitrogen, and then resuspended in 50 μL of methanol/chloroform (5:95 [vol/vol]). The samples were spotted at the base (2.5 cm from bottom) of thin-layer chromatography silica H plates, 2 cm apart, with constant drying by warm air to reduce the size of the spots. Chromatography was initiated by immersing the bottom (1 cm) of the plates in organic solvent (CHCl3/MetOH/NH4OH [4N], 9:7:2 [vol/vol/vol] for a total of 180 mL) in a vapor glass chamber. Chromatography was stopped when the yellow dye reached the top of the plate. The position of individual inositol lipids was determined by labeled standards. A 2×2-cm area of the thin gel corresponding to each phospholipid spot was scraped and counted in scintillation fluid.
PI3-K activity was blocked with the Penicillium-derived metabolite wortmannin. Wortmannin has been shown to inhibit the activity of PI3-K in a noncompetitive and irreversible fashion with an IC50 of <4 nmol/L.24 Serum-starved cells were incubated for 45 minutes with wortmannin (11 or 22 nmol/L) before exposure to PDGF-BB. The effect of PI3-K inhibition on actin polymerization after the addition of PDGF-BB was measured by rhodamine phalloidin assays (see above) and expressed as percent change in F-actin concentration relative to that of untreated cells.
Morphogenic Effects of EJ-Ras Transformation
NIH 3T3 clonal cell lines transfected with EJ-Ras cDNA, encoding an oncogenic mutant (valine substituted for glycine at amino acid 12) of the cellular Ha-Ras, appeared morphologically in a relaxed conformation when compared with control cells transfected with pCMVNeo vector only. Retracted, fusiform, and highly polarized cell bodies (with occasional long cytoplasmic extensions) having the tendency to cluster within foci (similar to stratified epithelium) were characteristic of the transformed phenotype. These morphological changes were associated with altered growth characteristics of EJ-Ras–transformed NIH 3T3 cells, as the growth and DNA synthesis activities of EJ-Ras–transformed cells were not inhibited at confluence or in medium containing low (0.5%) serum supplementation (data not shown). In contrast, Neo control cells formed monolayers of flattened and stressed cells and were growth-inhibited at confluence and/or in low serum.
Demonstration of EJ-Ras on Western Blots
To demonstrate further that these phenotypic changes observed in the transfected cells were associated with EJ-Ras expression, we assessed the overexpression of EJ-Ras protein by Western blot assay (Fig 1⇓). Lysates of two Ras-transformed cell lines (clones 5A2 and 5A6) and of EJ-Ras pool clones contained a band of p21ras immunoreactivity, not present in control cells (Neos), with an electrophoretic motility on 4% to 20% gradient SDS-PAGE that was slightly faster than the cellular p21ras present in all cell lines studied. This band was detectable both with anti–pan-p21ras and with anti–Ha-Ras–specific antibodies.
Changes in Actin Cytoskeleton in EJ-Ras Cells, With and Without PDGF-BB
Organization and distribution of the actin network in EJ-Ras–transformed cells was strikingly different from that in control cells (compare panels A and B of Fig 2⇓). When incubated in 0.5% FCS, Neo control cells contained multiple stress fibers, stable cell-spanning bundles of F-actin and associated regulatory proteins (stressed cells).17 These stress fibers were nearly absent in EJ-Ras–transformed clones; instead, F-actin was concentrated under the cell membrane, particularly at sites of membrane extension or ruffling.
PDGF-BB stimulation induced rapid redistribution of actin filaments in control cells, with loss of stress fibers from the cell center and concentration of F-actin under the plasma membrane, particularly in areas where membrane ruffling appeared prominent. In EJ-Ras–transformed cells, F-actin was already concentrated at the cell periphery, and further redistribution after PDGF-BB treatment could not be detected by confocal microscopy (data not shown).
F-actin quantification revealed that at baseline, control cells contained more polymerized actin than did EJ-Ras–transformed cells (Fig 3⇓). Thus, despite having a more motile phenotype, the EJ-Ras–transformed cells contained lower basal concentrations of F-actin (43% that of control cells). However, a marked actin polymerization response to PDGF-BB in EJ-Ras–transformed cells was observed, similar in extent and kinetics to that observed in control cells (Fig 3⇓). After 3 minutes in PDGF-BB, the concentration of polymerized actin peaked in all cell types studied, independent of the p21ras status, and was increased 60% over baseline in Neo control cells and 87% in EJ-Ras–transformed cells. Total actin, assessed by densitometric analysis of Western blots, was not affected by p21ras transformation (Fig 3⇓, inset), and was ≈70.3±7.5 μmol/L, or 1.4±0.2% of total cellular protein (mean±SEM). Calculations are based on estimated cell volume of 1923 μm3 as previously described.20
We concluded from this series of experiments that EJ-Ras transformation of NIH 3T3 cells induced marked morphological changes through reorganization of the actin cytoskeleton. Despite the effect of EJ-Ras on the actin cytoskeleton, we observed that the actin cytoskeleton of EJ-Ras–transformed cells was still responsive to PDGF-BB. To characterize the signaling pathways that regulate these morphological changes, we examined signaling reactions after exposure to PDGF-BB, starting with the tyrosine phosphorylation of the receptor itself.
PDGF-R Tyrosine Phosphorylation
In the absence of agonist, no phosphotyrosine immunoreactivity was detected on PDGF-R (Fig 4⇓, top) in either control or EJ-Ras–transformed cells. After agonist binding, tyrosine phosphorylation of the PDGF-R was detected within 1 minute; in control cells, PDGF-R phosphotyrosine immunoreactivity (density on Western blot developed by ECL) increased 16-fold over background, whereas 5A2 and 5A6 cells’ immunoreactivity at 1 minute increased only 6- and 4-fold, respectively. In control cells, continued PDGF-BB exposure resulted in a further marked increase in tyrosine phosphorylation; both the early response and the late increase of PDGF-R tyrosine phosphorylation were reduced in the EJ-Ras–transformed cell lines. After 20 minutes of exposure to PDGF-BB, PDGF-R phosphotyrosine immunoreactivity was increased 63-fold over baseline in control cells, whereas the activity of 5A2 and 5A6 cells increased only 10±5-fold over baseline. Total PDGF-R cellular content, assessed by Western blot, was similar in all cell types (data not shown).
Tyrosine Phosphorylation of PLCγ1
Because tyrosine phosphorylation of the PDGF-R in EJ-Ras cells was partially inhibited, we suspected that the phosphorylation of PLCγ1 might be impaired as well. Tyrosine phosphorylation of PLCγ1 is required for PIP2 hydrolysis in response to PDGF-BB and has been linked to the actin cytoskeletal response to growth factors.25 26 Western blot analysis of anti-PLCγ1 immunoprecipitates stained for phosphotyrosine residue revealed only mildly (30%) decreased tyrosine phosphorylation of PLCγ1 in EJ-Ras cells stimulated by PDGF-BB (30 μg/L) (Fig 4⇑, bottom). Actually, the level of tyrosine phosphorylation of PLCγ1 in EJ-Ras cells induced by 30 μg/L of PDGF-BB was between that induced by 10 and 30 μg/L PDGF-BB in control cells (Table⇓), and 10 μg/L of PDGF-BB was sufficient in control cells to induce IP3 production. Since the tyrosine phosphorylation of PLCγ1, which is required for PLCγ1 activity, was nearly intact in EJ-Ras cells, we studied the hydrolysis of PIP2 in these cells exposed to PDGF-BB.
PDGF Stimulation of IP3 Production Was Lacking in EJ-Ras–Transformed Cells
The time course of IP3 production measured by competitive binding assay revealed that PDGF-BB stimulated a 3.7-fold IP3 increase, peaking at 40 to 60 seconds in control NIH 3T3 cells (Table⇑). IP3 production was dependent on the dose of PDGF-BB, such that 3 μg/L of PDGF-BB stimulated no detectable IP3 increase, whereas 10 and 30 μg/L stimulated significant IP3 production (Table⇑). In contrast, in EJ-Ras–transformed NIH 3T3 cells, addition of PDGF-BB (30 μg/L) did not stimulate any IP3 production (Table⇑). Even after exposure of EJ-Ras–transformed cells to lithium (to block IP3 recycling), PDGF-BB–induced production of IP3 was still undetectable (data not shown). The absence of IP3 production in transformed cells is paralleled by the calcium transient response, which is present in control cells but absent in transformed cells (L.E.C., A.W.H., P.J.G.-C., R.W.T., unpublished data). Lack of IP3 increase did not result from constitutively elevated IP3 content in EJ-Ras–transformed cells (Table⇑). We next determined whether a lack of substrate (PIP2) could account for the absence of PDGF-stimulated IP3 production in EJ-Ras–transformed NIH 3T3 cells.
Phosphoinositide Content Is Similar in Control and EJ-Ras–Transformed Cells
The cellular content of PIP2, PIP, and PI was measured by thin-layer chromatography in control and EJ-Ras–transformed NIH 3T3 cells. Relative amounts of the phosphoinositides were similar in control and in EJ-Ras–transformed NIH 3T3 cells (Fig 5⇓). Phosphoinositide content was measured by scintillation counting of the scraped thin-layer chromatography gels corresponding to individual inositol phospholipid spots. Whether counts per minute values corresponding to the PIP and PIP2 spots were normalized to total protein or to the PI mass, no difference could be detected between control and EJ-Ras–transformed cells. Thus, the lack of PIP2 hydrolysis in PDGF-stimulated EJ-Ras–transformed cells was not a result of limited substrate availability.
Recombinant PLCγ1 Inhibition by EJ-Ras Cell Extracts
In spite of nearly normal tyrosine phosphorylation of PLCγ1 in PDGF-activated EJ-Ras–transformed cells, we found no detectable PLC activity in extracts from the Ras-transformed cells obtained before or after PDGF stimulation. This finding is consistent with our observation that the production of IP3 by EJ-Ras–transformed cells in response to PDGF-BB is negligible (Table⇑). Moreover, the activity of a purified recombinant PLCγ1 was markedly reduced upon the addition of cytosolic proteins from EJ-Ras–transformed cell extracts but not by the cytosolic proteins extracted from control cells (Fig 6⇓). As shown in Fig 6B⇓, 50 μg/mL of extracted protein was nearly able to block the activity of 14 nmol/L of recombinant PLCγ1. Together, these data support the concept that the cytosol of Ras-transformed cells contained an inhibitory factor (or factors) for PLCγ1 and that this inhibitor was responsible for the complete suppression of PLCγ1 activity in EJ-Ras–transformed cells.
PI3-K and Actin Response to PDGF
Since EJ-Ras transformation prevented PIP2 hydrolysis induced by PDGF, we investigated whether another aspect of inositol phospholipid turnover was involved with actin changes induced by PDGF. PI3-K, a key mediator of the signaling response orchestrated by activated PDGF-R, phosphorylates inositol lipids and contributes to the actin response to PDGF in nontransformed cells.7 27 We used a selective PI3-K inhibitor, wortmannin,24 to test whether PI3-K activation is required for PDGF stimulation of actin changes in EJ-Ras–transformed cells. Pretreatment of EJ-Ras–transformed cells with wortmannin completely abolished the polymerization of actin induced by PDGF-BB (Fig 7⇓). Indeed, exposure of both control cells and EJ-Ras–transformed cells to wortmannin led to rapid depolymerization of actin filaments, a response quite the opposite of that seen in cells not treated with wortmannin. Even higher concentrations of PDGF-BB (up to 100 μg/L) did not overcome wortmannin inhibition (Fig 7⇓). Using immunofluorescence microscopy, we confirmed the presence of PI3-K within the cytoplasm of control and Ras-transformed cells and its concentration within the ruffling area of the plasma membrane (data not shown). After treatment with wortmannin and PDGF, rhodamine phalloidin microscopy showed that what little F-actin remained was organized in spotty clusters; these cells were not rounded, however, and resembled untreated cells except that ruffles were not present after exposure to PDGF (data not shown). These experiments suggest that unlike PLCγ1, PI3-K plays an important role in the regulation of the actin cytoskeleton and morphological changes observed in NIH 3T3 cells transformed with EJ-Ras.
Actin Organization in EJ-Ras–Transformed Cells
F-actin concentration (normalized for total protein) is reduced in the EJ-Ras–transformed cell lines, consistent with the observation of Rao et al,28 although total actin content does not differ between transformed and control cells. The increased basal and PDGF-stimulated motility of the transformed cells therefore does not correlate with the cellular concentration of F-actin. Rather, it is the distribution of F-actin, and in particular its local concentration within membrane ruffles, which correlates best with the dynamic morphological changes associated with the transformed phenotype and with PDGF-BB stimulation.
The EJ-Ras–transformed phenotype suggests a crucial role for Rac in the organization of the actin cytoskeleton of these cells8 9 10 29 30 ; the morphology of EJ-Ras–transformed cells is similar to that obtained with a dominant positive Rac mutant microinjected in fibroblasts.9 10 The involvement of Rac in the transformed phenotype is further suggested by the response to PI3-K inhibition, since PI3-K controls the association of Rac with GTP.13 Wortmannin not only inhibits PI3-K and blocks the expected actin response to PDGF of control and EJ-Ras–transformed cells but actually reverses the response to PDGF. In wortmannin-treated EJ-Ras and control cells, PDGF exposure leads to actin depolymerization, suggesting that the regulation of balance between monomeric versus filamentous actin by PDGF is complex and involves concurrent production and disruption of actin filamentous structures, with production under the influence of PI3-K.
Little is known about reactions downstream from small GTP-binding proteins that connect these proteins to the actin cytoskeleton. Several actin-binding proteins are regulated by inositol phospholipids in their interaction with actin.27 Profilin is an essential actin-binding protein that regulates actin monomers and filament stability and binds to polyphosphoinositides (PIP and PIP2).31 The distribution of F-actin beneath the plasma membrane and within the cortical cytoskeleton of EJ-Ras–transformed cells is reminiscent of the distribution of actin filaments in cells overexpressing profilin20 or microinjected with profilin32 and of actin filaments that have incorporated microinjected G-actin subunits having a high affinity for profilin.33
Thus, profilin may represent a downstream effector protein for the p21ras pathway,27 directly regulating the organization of the actin cytoskeleton. Such a hypothesis is also supported by experiments performed in yeast, showing that overexpression of profilin can rescue S. cerevisiae mutants null for the C-terminal of the p21ras-regulated adenylate cyclase associated protein.34 Adenylate cyclase–associated protein C-terminal null cells display altered morphology that can be corrected by profilin overexpression. Thus, the ability of profilin to stabilize cortical actin filaments may contribute to the actin cytoskeleton organization in EJ-Ras cells. Importantly, the effect of profilin is unlikely to be related to its ability to inhibit PLCγ1,26 since this enzyme is inactive in EJ-Ras–transformed cells (Fig 7⇑).
Other actin regulatory proteins have been implicated in the genesis of the EJ-Ras–transformed actin phenotype. Overexpression of tropomyosin, whose binding to actin stabilizes filaments in vivo, has been shown to suppress the neoplastic growth of Ras-transformed cells, possibly by causing reversion to a phenotype with more F-actin.35 Flat revertant clones of EJ-Ras–transformed NIH 3T3 cells have been found to contain a gelsolin mutant with increased affinity for polyphosphoinositides, resulting in stronger inhibition of PLCγ1 in vitro.36 37 Competitive inhibition by polyphosphoinositides of gelsolin’s capping function for actin might decrease the actin critical concentration in these revertants, resulting in more actin polymerization and in a flat revertant cytoskeletal phenotype.36 37 38 However, the mechanism whereby EJ-Ras might affect filament capping remains to be characterized.
Intact Actin Response to PDGF in EJ-Ras–Transformed Cells
The p21ras mutations, such as the one found in the EJ-Ras isoform, generate a constitutive GTP-bound conformation of the small GTP nucleotide triphosphatase14 and represent dominant mutations that substantially alter the biology of the affected cells in a concentration-dependent fashion.39 For example, transforming p21ras isoforms alter normal growth regulation, such that cells do not become quiescent (G0) at confluence or in the presence of sparse nutrient supplies. EJ-Ras–transformed cells are also remarkable for their decreased adhesion leading to relaxed morphology40 and reduced tyrosine kinase response to growth factors like PDGF. The morphology of these cells strongly resembles that of fibroblasts attached to an extracellular matrix that has been mechanically relaxed.17
In this context, we had anticipated that the actin response to growth factors such as PDGF would be markedly altered, if not absent, as a result of EJ-Ras transformation. Although we confirmed that EJ-Ras–transformed cells have increased motile activity in the absence of agonist, we found that, against expectations, the actin response to PDGF is essentially intact in EJ-Ras–transformed cells.
Actin Response to PDGF Occurs Despite PLCγ1 Inhibition in EJ-Ras Cells
Two phospholipid regulatory enzymes, PI3-K and PLCγ1, are targets for the tyrosine kinase of the PDGF-R and contribute to the actin response to PDGF.7 41 Although we confirmed that EJ-Ras transformation reduced the tyrosine phosphorylation of the PDGF-R itself,15 16 42 43 the tyrosine phosphorylation of PLCγ1 was nearly intact.44 In spite of normal phosphorylation of PLCγ1, PIP2 hydrolysis in EJ-Ras cells stimulated by PDGF was undetectable (Table⇑). Although we did not rule out the possibility that qualitative alterations of PLCγ1 phosphorylation may have occurred (four tyrosine residues can be phosphorylated on PLCγ1)45 as a result of EJ-Ras transformation, quantitatively, the net tyrosine phosphorylation of this substrate could not account for the lack of PLC activity in the transformed cells.
Instead, the cytosol of EJ-Ras–transformed cells contained an inhibitor (or inhibitors) that suppressed the activity of cytoplasmic PLC and prevented the hydrolysis of PIP2 by recombinant PLCγ1 added to the extracts. Inhibition of PLCγ1 in EJ-Ras–transformed cells was not due to profilin, because profilin inhibits only the unphosphorylated conformation of PLCγ1 and not tyrosine-phosphorylated PLCγ1.26 Although we are presently working on identifying the PLCγ1 inhibitor induced by EJ-Ras expression, our data are consistent with a catalytic mechanism of inactivation (Fig 7⇑).
PI3-K Is Required for the Cytoskeletal Response to PDGF
There is growing evidence that PI3-K plays a pivotal role in the motile (and mitogenic) responses of cells exposed to growth factors.46 Indeed, we found that an intact PI3-K function was necessary to generate a normal actin response to PDGF in EJ-Ras–transformed (and control) cells. Wortmannin, an inhibitor for PI3-K that is specific when used at low nanomolar concentrations,24 47 efficiently prevents PDGF-stimulated polymerization of the actin cytoskeleton. PI3-K is present within ruffles of PDGF-activated NIH 3T3 cells48 49 50 and in EJ-Ras–transformed cells, with and without growth factor stimulation (data not shown).
Four mechanisms may contribute to the association of PI3-K with the plasma membrane. First, the SH2 domain of the p85 docking subunit of PI3-K binds to tyrosine-phosphorylated residues 708 and 719 of the activated PDGF-R.7 Second, GTP-bound p21ras that is membrane associated (through its farnesyl group) binds to PI3-K.51 The latter mechanism is likely to play an important role in the association of PI3-K with the ruffles of EJ-Ras–transformed cells, even in the absence of growth factor stimulation. In support of this idea, farnesyl-transferase inhibitors reverse the phenotype of cells transformed with EJ-Ras to flattened cells displaying normal actin stress fibers.52 Third, PI3-K may bind directly to its phospholipid substrates in the membrane. Fourth, PI3-K stabilizes the association of Rac with GTP.13 In turn, GTP-Rac may stabilize the interaction between p21ras and PI3-K, or alternatively, Rac may promote the generation of oxygen reactive species, through its interaction with the NADPH-oxidase complex,53 which could affect the stabilization of actin filaments within ruffles.
These findings may be of importance for atherosclerosis and restenosis. The intracellular pathways regulating cell motility are affected by context: the migration of smooth muscle cells to the intima is highly dependent on the activity of PLCγ1.6 41 However, p21ras mutations leading to a p21ras-on state of these cells can block the PLC pathway. In such cells, the PI3-K pathway becomes essential for the actin reorganization in response to growth factors. Although our understanding of the role of p21ras in proliferation and migration of vascular cells is still scanty, it is likely that modulation of this pathway will become an important therapeutic option for the management of plaque formation and restenosis.
Selected Abbreviations and Acronyms
|Neo cell||=||neomycin-resistant cell|
|PDGF||=||platelet-derived growth factor|
|SSLV||=||small synthetic lipid vesicle|
This study was supported by grants from the American Heart Association, Maryland Affiliate, Inc (Grant-in-Aid); from Syntex; from the Bernard A. and Rebecca S. Bernard Foundation; and from Friends of Bobbie Burnett. Dr Heldman was the recipient of a 1993 and 1994 Research Fellowship from the American Heart Association, Maryland Affiliate, Inc. Dr Goldschmidt-Clermont was selected as a Syntex Scholar in 1992.
- Received August 3, 1995.
- Accepted November 6, 1995.
- © 1996 American Heart Association, Inc.
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