Effects of Platelet-derived Growth Factor on Phosphorylation of the ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 264, No. 16, Issue of June 5, pp. 9204-9209,1989 Printed in U.S. A.

0 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Effects of Platelet-derived Growth Factor on Phosphorylationof the Epidermal Growth Factor Receptor in Human Skin Fibroblasts* (Received for publication, October 14,1988)

Stuart J. Decker and Paul Harris From the Rockefeller University, New York, New York 10021

Heterologousregulation of theepidermalgrowth receptor systems may be co-ordinated in some manner. For factor (EGF) receptor by platelet-derived growth fac- some cells PDGF appears to be a “competence” factor which tor (PDGF) was studied in FS4 human skin fibroblasts. sensitizes cells to themitogenic effects of secondary mitogens The additionof PDGF to FS4 cells inhibited high affin-such as EGF(12,13).Exposure to PDGFinhibits high affinity ity bindingof ‘*‘I-EGF and stimulated phosphorylation binding of Iz5I-EGFby several types of fibroblasts (14,15). In of the EGF receptor. Phosphopeptide analysis by high human lung fibroblasts, PDGF-dependent inhibition of EGF performanceliquidchromatographyrevealedthat binding correlates with PDGF-induced phosphorylation of PDGF treatmentof cells increased phosphorylationat threonine 654 of the EGF receptor and with inhibition of several distinct sites of the EGF receptor. However, PDGF did not stimulate phosphorylation of threonine EGF receptor autophosphorylation on tyrosine residues (16, 654, a residue previously shown to be phosphorylated 17). Since PDGF has been shown to induce release of Ca2+ from intracellular stores and increase levels of diacylglycerol when protein kinase C is activated. The tumor promoter 12-0-tetradecanoylphorbol-13-acetate(TPA) (91, PDGF-induced phosphorylation of threonine 654 could also stimulated phosphorylation of the same peptides be due to activation of protein kinase C by these effectors. The effects of tumor promoting phorbol diesters on EGF from the EGF receptor as PDGF, and,inaddition, induced phosphorylationof threonine 654. TPA inhib- receptor metabolism are,in many ways, similar to those ited both high and low affinity ’“1-EGF binding by reported for PDGF in human lung fibroblasts. Treatment of (TPA) inhibits bindthese cells. PDGF treatment of cells had no effect on cells with 12-0-tetradecanoyl-13-acetate ing of ‘251-EGF(1,18)as well as the EGF-dependent tyrosine EGF-dependent, tyrosine-specific autophosphorylation of the receptor, whereas TPA treatment was in- kinase activity of the EGF receptor (19-22). TPA has also hibitory. TPA, but not PDGF, stimulated phosphoryl- been shown to stimulate protein kinaseC-mediated phosphoation of a M, = 80,000 protein, known to abesubstrate rylation of threonine 654 of the EGFreceptor (22, 23). In this for protein kinase C, even though PDGF appeared to report, we show that the effects of PDGF and TPAon EGF mediate breakdown of phosphoinositides. These data receptor metabolism in human skin fibroblasts are different suggest that regulation of EGF receptor function by from results previously reported for human lung fibroblasts PDGF and TPA are distinct in these cells, even though (16, 17). some elements of regulation are shared. The results differ from those previously reporteda human for lung EXPERIMENTALPROCEDURES fibroblast isolate, indicating that cell type-specific difMaterials-Tosylpbenylalanyl chloromethyl ketone (TPCK)ferences may exist in metabolism of the EGF receptor.

treated trypsin was obtained from Cooper Laboratories. TPA and 4a-phorbol were obtained from LC Biochemicals. Receptor grade epidermal growthfactor was fromCollaborative Research (Waltham, MA), andpurified human PDGFwas a generous gift from Dr. Thomas Epidermal growth factor (EGF)’ and platelet-derived Deuel, Washington University or was from R and D Systems, Inc., growth factor (PDGF) are potent mitogens for a number of Minneapolis,MN.lZ5I-EGFandcarrier-free[32P]orthophosphate different cell types. Both of these ligands induce cell division were from Du Pont-New EnglandNuclear. 1251-ProteinA, [2-3H]myoinositol and [3H]inositol polyphosphate standards were from Amerthrough bindingto specific cell surface receptors. Upon ligand sham Corp. Antiserum against the rat80,000 protein was generously binding, these receptors initiate a series of events that cu- provided by Dr. JamesWang, Rockefeller University, purified protein mulatively lead to cell division (1,2). Both receptors possess kinase C from calf brain was a kind gift from Antony Rosen, Rocketyrosine-specific protein kinase activity which is dependent feller University, and monoclonal anti-EGF receptor antibody 528 on ligand binding (3-5). EGF and PDGFhave been shown to was kindly provided by John Mendelsobn, Memorial-Sloan Kettering cause transient elevation of c-fos and c-myc transcription (6- Cancer Center. Cell Culture-Human foreskin fibroblasts FS4 (kindlyprovided by 8), and bothincrease turnover of phosphoinositides in certain J a n Vilcek, New York University and Pravin Sehgal, Rockefeller cell types (9-11). University) were grown in Dulbecco’s modified essentialmedium Several reports suggest that the PDGF receptor and EGF supplemented with 10% fetal calf serum. Cells were used 5-7 days after reaching confluence. Analysis of lZ5I-EGFBinding-Confluent FS4 cells in multiwell * This work was supported by Grant CA37754 from the National Institutes of Health. The costs of publication of this article were plates were washed once with binding buffer (Dulbecco’s modified defrayed in part by the payment of page charges. This article must Eagle’s medium with 50 mM HEPES, pH 7.4, and 1 mg/ml bovine buffer withorwithout therefore be hereby marked “aduertisement” in accordance with 18 serum albumin) and incubated in binding PDGF or TPAfor various periods of time. Indicated concentrations U.S.C. Section 1734 solely to indicate thisfact. of lZ5I-EGFwere then added, and thecells were incubated a t 4 “C for ‘ T h e abbreviations usedare: EGF,epidermal growthfactor; PDGF, platelet-derivedgrowth factor; TPA, 12-0-tetradecanoylphor- 4 h. Cells were washed four times with ice-cold binding buffer and bol-13-acetate;HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesul-solubilized with 0.5 ml of 0.5 M NaOH. After 5 min the radioactivity fonic acid HPLC, high performance liquid chromatography; TPCK, in the lysates was quantitated by liquid scintillation counting. Nonspecific binding was determined by parallel incubations containing L-1-tosylamido-2-pbenylethyl chloromethyl ketone.

9204

9205

Effects of PDGF on EGF Receptor Metabolism 500 ng/ml cold EGF. Each determinationwas performed in duplicate. Immunoprecipitation of in Vivo Phosphorylated EGF Receptor and M, = 80,000 Protein-FS4 fibroblasts were labeled with 32Pfor 16 h in phosphate-free Dulbecco's modifiedEagle's medium supplemented with 0.5% fetal calf serum and 2 mCi of 32P/ml. Labeled cells were lysed directly in hot 2% sodium dodecyl sulfate and heated at 100 "C for 5 min. Lysates were diluted in RIPA buffer (20) without sodium dodecyl sulfate to an sodium dodecyl sulfate concentration of 0.1% and immunoprecipitated with polyclonal antisera prepared against the EGF receptor as previously described (20) or with rabbit antiserum against the M, = 80,000 protein shown to be a substrate for protein kinase C (25). Quantitation of immunoprecipitated =P-labeled proteins was done by excising bands from gels, placing the gel pieces in water, and counting Cerenkov radiation in a liquid scintillation counter. Preparation of Tryptic Phosphopeptides-Immunoprecipitated 32Plabeled EGF receptor was run on sodium dodecyl sulfate polyacrylamide gels and located within the gel by autoradiography. Gel slices containing EGF receptor were washed for 15 min in 5 ml of water, followed by 5 ml of200 mM ammonium bicarbonate, pH 8.5, and incubated in 500 p1 of 200 mM ammonium bicarbonate containing 30 pg of TPCK-trypsin for 5 h at 37 "C, at which time another 30 pg of trypsin was added for 16 h. This treatment resulted in the release of 90-95% of the counts from the gel slices. The digests were lyophilized prior to two-dimensional thin layer (20) or HPLC analysis. Phosphpeptide Mapping by Reverse-phase HPLC-Tryptic phosphopeptides were separated by reverse-phase HPLC on a Cle column equilibrated with 0.05% trifluoroacetic acid in water. The 32P-labeled phosphopeptides were eluted from the column with a linear gradient of acetonitrile (0-60%) in 60 min a t a flow rate of 1 ml/min. Analysis of Inositol Phosphates-Five-day postconfluent cultures of FS4 cells in 35-mm dishes were labeled for 18-20 h with 10 pCi/ ml [3H]inositol added directly to the normal growth medium. This medium was then replaced with Dulbecco's modified Eagle's medium containing 10 mM LiCl and 0.5% bovine serum albumin and cells were incubated for 10 min at which time additions were made for the indicated periods. Incubations were terminated by the addition of 250 yl of ice-cold 5 M perchloric acid containing 1 mM EDTA and placed on ice for 5 min. The extracts were neutralized with 50 pl of 2.5 M K2C03 andplaced on ice for at least 5 min before analysis of HPLC on a Whatman Partisil SAX 10 column (0.46 X 25 cm) equilibrated with water. Samples were loaded on the column and inositol phosphates eluted using a modification of the method of Dean and Moyer (24). Following addition of samples the column was washed with water for 5 min. The gradients used were 0-0.1 M ammonium phosphate, pH 3.8, with phosphoric acid for 15 min, 0.1-0.3 M ammonium phosphate for 10 min, and 0.3-1.0 M ammonium phosphate for 25 min. Authentictritium-labeled inositol-1-phosphate, inositol-l,4standards were used to caliphosphate, and inositol-1,3,4-phosphate brate the column. In this system, these standards eluted at about 21, 38-39, and 50 min, respectively. Peptide Synthesis and Phosphorylation-The peptide Lys-ArgThr-Leu-Arg was synthesized by the Rockefeller University Sequencing Facility. The peptide was purified by HPLC and phosphorylated by protein kinase C (purified protein kinase C was kindly provided by Anthony Rosen, Rockefeller University) according to Hunter et al. (23). The phosphorylated peptide was isolated by electrophoresis at pH 8.9 (1%(NH4)&03) at500 V for 45 min. Binding of Monoclonal EGF Receptor Antibodyto FS4 Cells-Fiveto 7-day-old confluent cultures of FS4 cells in 1.5-cm wellswere washed once with ice-cold Dulbecco's modified Eagle's medium containing 50 mM HEPES, pH 7.4, and 1.0% bovine serum albumin (binding buffer). 0.2 ml of binding buffer containing 10 pgof anti-

TABLE I Effects of EGF and PDGF on incorporation of PHIthymidine by FS4 celk Addition

[3H]thymidineincorporation"

cpmlmg protein

None 4,308 10 ng/ml EGF 28,438 32 ng/ml PDGF 26,781 10 ng/ml EGF + 32 ng/ml PDGF 36,725 Confluent cells were incubated in serum free medium for 12 h, prior to incubation with the indicated additions for 24 h. Incorporation of [3H]thymidine was then measured as described in the text.

\

1

a

A

-

3

?Po $0

Y

0

I

$0

I

I

90 120 150 Bound (p Molar)

n

I

I

180

210

i

240

B

Control

k, I

TPA

2

3

a

4

Time (hours)

FIG. 1. PDGF and TPA-mediated inhibition of "'I-EGF binding by FS4 cells. In A, Scatchard analysis of binding of '"1EGF to control FS4 cells (circles), cells treated for 20 min at 37 "C with 32 ng/ml PDGF (triangles), or with 25 ng/ml TPA (sqmres) is shown. Cellswere incubated with various concentrations (0.11-20 ng/ml) of '"I-EGF and thespecific binding determined. Points shown represent the mean of duplicate samples. Each experiment was repeated three times with essentially the same results. In E , the effects of PDGF or TPA treatment of cells on the time course of '"I-EGF binding by FS4 human skin fibroblasts are shown. Specific binding of '"I-EGF (0.66 ng/ml) by control cells (circles),cells incubated with 32 ng/ml PDGF (squares), or with 25 ng/ml TPA (triangles) was determined at various times following 'T-EGF addition. Cells were incubated with PDGF or TPA for 20 min prior to T - E G F addition. Binding was performed as described under "Experimental Procedures." EGF receptor monoclonal antibody 528 (26) was then added, and cells were incubated a t 0 "C for 4 h. Cells were washed three times with 0.5 ml of ice-cold binding buffer and 0.2 ml of binding buffer containing 200 ng of lZI-protein A (1 pCi) was added. After incubation at 0 "C for 60 min, cells were washed three times with 0.5 ml of binding buffer and lysed by the addition of 0.5 ml of0.5 N NaOH. Radioactivity in each sample was determined by liquid scintillation counting. For determination of nonspecific background counts, cells

PDGF Effects of

9206

on EGF Receptor Metabolism

TABLE I1 Effects of TPA, PDGF, and EGFon binding of EGF receptor monoclonal antibody 528 to FS4 cells. Binding of antibody and 1Z5-I-proteinA to FS4 cells were performed as described under “Experimental Procedures.” The data presented are the mean and standard error of resultsobtained from three separate experiments. Treatment

?6 binding to control

Control 25 ng/ml TPA, 20 min 32 ng/ml PDGF, 20 min 100 ng/ml EGF, 40 min

100 84.1 f 7.7 95.3 f 4.1 32.7 f 5.8

-200 ” ”

- 68

-

43

FIG. 2. Phosphorylation of the EGF receptor in FS4 cells. FS4 cells were labeled in phosphate-free medium with 32Pfor 16 h. EGF receptors were immunoprecipitated from 32P-labeled control cells, and from cells treated for 20 min with 50 ng/ml EGF, 32 ng/ml PDGF or TPA (25 ng/ml). The cells were labeled and EGF receptors isolated by immunoprecipitation as described under “Experimental Procedures.” The mobilities of known standards (X10-3) are shown in the right-hand margin. This experiment was performed six times and a typical result is shown. were treated as above except nonimmune mouse IgG was included in place of antibody 528. Background was 10-15%of binding to controls. Two-dimensional thin layer phosphopeptide analysis, phosphoamino acid analysis, assay of [3H]thymidineincorporation, and SDSpolyacrylamide gel electrophoresis were performed as previously described (20). RESULTS

The data presented are from experiments examining the effects of PDGF on metabolism of the EGF receptor in FS4 human skin fibroblasts (27), however, similar results were found with three other independent human foreskin fibroblast isolates. Both PDGF and EGF were strongly mitogenic for quiescent FS4 cells as judged by their ability to increase the incorporation of [3H]thymidine into acid precipitable counts (Table I). PDGF and EGF added together were somewhat

FIG. 3. Two-dimensional thin layer analysisof tryptic phosphopeptides of EGF receptors fromFS4 cells. Phosphopeptides maps of EGF receptor from control cells ( A ) , cells treated with 32 ng/ml PDGF for 20 min ( B ) ,and cells treated with 25 ng/ml TPA for 20 min ( C ) are shown. The basic peptide containing phosphorylated threonine 654 from TPA-treated cells is designated peptide ( 2 ) . The EGF receptors were immunoprecipitated, trypsin treated, and analyzed in two dimensions as described in the text. Electrophoresis was in the horizontal dimension (cathode on the left). Equal amounts of tryptic peptides were run for each sample, and these experiments were performed three times with similar results.

more effective than eitheralone. Comparison of PDGF and TPA Mediated Inhibition of lZ5IEGF Binding by FS4 Cells-Since PDGF and TPA have been shown to inhibit EGF binding by a number of cell types (1, 14,15, 18),we compared their effects on EGFbinding by FS4 cells. Scatchard analysis (Fig. 1A) ofI2’I-EGF binding revealed two classes of binding sites in normal FS4cells; about 27,300 f 4,536 high affinity binding sites/cell (Kd = 5.1 zk 0.75 X 10”’ M) andabout 135,000 k 17,847 low affinity binding sites/cell ( K d = 7.5 zk 0.54 X lo-’ M). The observed PDGF-dependent inhibitionof EGF binding was due primarily to loss of the high affinity EGF-binding sites with the remaining low affinity sites having a Kd of 8.0 f 0.9 X lo-’ M (125,902 f 14,548 binding sites/cell). TPA treatment caused an inhibition of both high and low affinity EGF binding leaving one class of very low affinity binding sites with an apparent Kd of 4.1 0.8 X lo-’ M (71,999 9,693 binding sites/cell). Both PDGF and TPA caused prolonged inhibition of binding of ’251-EGFand the inhibition by TPA was more

+

+

Effects of PDGF on EGF Receptor Metabolism

FIG. 4. Confirmation that peptide z contains phosphorylated threonine 654. Peptide z was scraped from several thin layer plates on which tryptic digests of EGF receptor from TPA-treated FS4 cells had been run (as Fig. 3C). Based on previous studies and its characteristic mobility in two-dimensional analysis peptide z was believed to be Lys-Arg-ThrP-Leu-Argcontaining phosphorylated threonine 654. Authentic Lys-Arg-ThrP-Leu-Argwas synthesized as described in the text. Peptide z and the authentic pentapeptide were subjected to two-dimensional thin layer analysis as described under “Experimental Procedures.” In A, 100 cpm of peptide z was run, in B , 200 cpm of authentic peptide, and in C, 100 cpm of peptide z was run together with 200 cpm of authentic peptide. The origins are marked 0. Electrophoresis was in the horizontal dimension (cathode on the left).Plates were exposed for 48 h a t -70 “Cwith intensifying screens.

3001

1

-Q-

9207

,200

-97

-68

Conlml PDGF

FIG.6. Phosphorylation of the M, = 80,000 protein. FS4 cells were labeled with ‘*P and M,= 80,000 protein immunoprecipitated as described in the text. Immunoprecipitates from control cells (first lane on the left), cells treated for 20 min with 32 ng/ml PDGF (center l a n e ) , or with 25 ng/ml TPA (right l a n e ) are shown. The mobilities of known standards (X10-3) are shown in the right-hand margin. This experiment was performed twice with essentially identical results.

300

200

0

10

20

30

40

Acetonitrile concentration(“70)

FIG. 5. Analysis of EGF receptor tryptic phosphopeptides by HPLC. In vivo 32P-phosphate-labeled EGF receptors were immunoprecipitated from control, PDGF (32 ng/ml) treated, and TPA (25 ng/ml)-treated FS4 cells. Gel slices containing the receptors were trypsintreated and resulting phosphopeptides resolved on a Cn reverse-phase column as described in the text. Equal amounts of tryptic peptides were analyzed for each type of sample. These experiments were repeated four times with similar results and the data shown are from a typical experiment.

pronounced (Fig. 1B). These observed changes in EGF binding by PDGF were apparently not attributable to loss of cell surface EGF receptors as the specific binding of anti-EGF receptor monoclonal antibody 528 (26) was not significantly changed (Table 11).TPA treatment caused a 10-20% decrease in the amount antibody specifically boundto thecell surface. PDGF-stimulated Phosphorylation of the EGF Receptor in FS4 Cells-In addition to inhibiting EGF binding, PDGF has been shownto stimulate phosphorylation of the EGFreceptor (16, 17). We also found a consistent 30-40% increase in 32P-

incorporation into the EGF receptor when FS4 cells were treated with PDGF (Fig. 2). Using WI-38 lung fibroblasts, it has been reported that residue threonine 654of the EGF receptor is phosphorylated when cellsare treatedwith PDGF or TPA (16, 17, 22).To examine this possibility in FS4 cells, tryptic digests of EGF receptor from control, PDGF treated, and TPA-treated cells were subjected to two-dimensional thin layer analysis (Fig. 3). Surprisingly, no PDGF-stimulated phosphorylation of the peptide containing threonine 654 was detected in cellstreated with PDGF (Fig. 3B), although TPAstimulated phosphorylation of this phospho-threonine-containing peptide was easily discernable (peptide z in Fig. 3C). Peptide z was shown to be the peptide Lys-Arg-ThrP-LeuArg containing phosphorylated threonine 654 (residues 652656 of the EGF receptor) by several criteria in addition to its distinctive position in two-dimensional analysis. First, peptide z was found to comigrate in two-dimensional thin layer analysis with synthetic Lys-Arg-Thr-Leu-Arg pentapeptide which had been phosphorylated in vitro by protein kinase C (Fig. 4). Secondly, TPA failed to stimulate phosphorylation of a peptide migrating as peptide z in tryptic digests of EGF receptor-containing alanine at position 654 which had been expressed in 3T3 cells (data not shown). The peptide map in Fig. 3B is for EGF receptor from cellstreated for 20 min with PDGF. However, treatment with PDGF for 2,5, and 60 min also did not result in detectable phosphorylation of threonine 654 (not shown). These data suggested that PDGF-stimulated phosphoryla-

Effects of PDGF on EGF Receptor Metabolism

9208

TABLE I11 Effects of PDGF on accumulationof inositol monophosphate ( I m p , ) , inositol bisphosphate (ImP2), andinositol trisphosphate (InsPd in FS4 cells FS4 cells were labeled for 20 h with 10 pCi/ml [3H]inosit~l and measurements of inositol phosphate levels were performed as described under “ExperimentalProcedures.” Cells were treated with vehicle or PDGF for 3 min prior to analysis. The data presented are the mean and standard error from three separate experiments. InsP,

Addition

Control 32 ng/ml PDGF

InsP,

cpm

% Control

3315 -t 436 5990 +- 719

500 100 788 181

cpm

f 103 f 137

InsP3 76 Control

205 100 157

cpm

% Control

100f 37 402 f 45

196

FIG. 7. Analysis of acid stable phosphoamino acids from S2P-labeledEGF receptors from FS4 cells. Phosphoamino acid analysis of in uiuo labeled EGF receptor from control cells ( A ) ,from cells treated with 10 ng/ ml EGF for 10 min ( E ) ,with 32 ng/ml PDGF for 20 min followed by 10 ng/ml EGF for 10 min (C), or with 25 ng/ ml TPA for 20 min followed by 10 ng/ml EGF for 10 min (D)are shown. EGF receptors were immunoprecipitated and phosphoamino acid analysis performed as described in the text. The position of phosphotyrosine ( p Y ) , phosphothreonine (~5”).and phosphoserine (DS)standardsare shown in lane E superimposed ona sample autoradiogram. Equal amounts of -hydrolyzed’receptor were analyzed. These results were reproduced in three experiments. ”

tion of the EGF receptor from FS4 cells occurred at sites other than threonine 654. Therefore, phosphopeptides obtained from tryptic digestion of EGF receptors were applied to a reverse-phase HPLC column foradditional characterization. The siteson the EGFreceptor phosphorylated in control cells included two major phosphopeptides eluting at about 23% acetonitrile and 30% acetonitrile (Fig. 5). The phosphopeptides obtained from PDGF-treated EGF receptors eluted in a similar pattern butwith increased radioactivity associated with the majorpeaks. The phosphopeptide map of TPAtreated EGF receptors revealed increased phosphorylation at sites common withPDGF-treated EGF receptors. In addition, a peak containing phosphorylated threonine 654 eluting at about 3% acetonitrile was found only in EGF receptor from TPA-treated cells. Authentic Lys-Arg-ThrP-Leu-Arg coeluted with this peak. These results suggested that PDGF treatmentof FS4 cells may not lead to activation of protein kinase C to the same extent as has been reported for certain other cell types (2830). To further investigate this possibility, we examined the effects of PDGF and TPA on phosphorylation of a M , = 80,000 protein first described by Rozengurt et al. (25) which is known to be a substrate for protein kinase C.We found that TPA treatment of FS4 cells caused an approximate 3fold increase in incorporation of 32P into this M , = 80,000 protein. However, PDGF treatment of cells waswithout effect (Fig. 6). Despite our inability to demonstrate PDGF-dependent activation of protein kinase C, PDGF treatment of FS4 cells caused some increase in phosphoinositide turnover as judged by its capacity to stimulate accumulation of inositol phosphates in these cells (Table 111). Effects of PDGF on in Vivo Tyrosine Phosphorylationof the EGF Receptor-Acid stable phosphoamino acid analyses were performed on EGF receptors from control FS4 cells and from cells treated with EGF, with PDGF followed by EGF, or with TPA followed by EGF (Fig. 7). EGF receptor from control cells contained phosphoserine and phosphothreonine, with no detectable phosphotyrosine (Fig. 7A). EGF treatment stimulated tyrosine phosphorylation of the receptor as previously reported (5,31). When FS4 cells were treated with TPA prior

”

to EGF, the EGF-stimulated increase in tyrosine phosphorylation was inhibited. In contrast, prior treatment of FS4 cells with PDGF, followed by EGF, had no effect on EGF-stimulated tyrosine-specific autophosphorylation of the EGF receptor. DISCUSSION

In this report we have examined the effects of PDGF on metabolism of the EGF receptor in FS4 human foreskin fibroblasts. Our data indicate that certain aspects of PDGFmediated regulation of EGF receptor function in FS4 cells are distinct from those previously reported for WI-38 human lung fibroblast cells (16, 17). As previously shown for a number of other cell types (l), FS4 cells exhibit high and low affinity EGF binding, and PDGF treatment results in the loss of the high affinity binding component. The addition of TPA to cultures of FS4 cells also decreases the binding of lz5I-EGF,but unlike PDGF, effects both high and low affinity binding. These data reveal that EGF binding can be modulated to varying degrees by PDGF and TPA in skin fibroblasts. In contrast, PDGF and TPAmediated regulation of EGF receptor binding affinity in WI38 lung fibroblasts were similar with both effectors preferentially reducing highaffinity binding (17). Inhibition of EGF binding by PDGF and TPA has been shown to correlate wiih increased phosphorylation of the EGF receptor (1, 16-18). PDGF and TPA also stimulate phosphorylation of the EGF receptor in FS4 cells but in qualitatively different manners. A comparison of phosphopeptide maps from PDGF and TPA-treated EGF receptor showed that both increase phosphorylation of several endogenously phosphorylated peptides. OnlyTPA induced phosphorylation of threonine 654. These results contrast with results reported using WI-38 cells (16,22), where in addition to increasing phosphorylation of endogenously phosphorylated peptides, both PDGF and TPA induce phosphorylation of threonine 654. Our data suggest that heterologous regulation of EGF receptor binding affinity can occur independently of phosphorylation of threonine 654. It appears that EGF binding may be regulated by EGF receptor phosphorylation through at least the

Effects of PDGF on EGF Receptor Metabolism following two mechanisms: 1) by increased phosphorylation of regions of the EGF receptor represented by the endogenously phosphorylated peptides, and 2) by phosphorylation of threonine 654. These two mechanisms may act in an additive fashion. PDGF has been shown to activate protein kinase C in a number of cell types (25,29,30). Since threonine 654 is known to be phosphorylated by proteinkinaseC (19), our data suggest that PDGF does not appreciably activateprotein kinase C in FS4 cells. This idea is supported by the inability of PDGF to stimulate phosphorylation of an M , = 80,000 protein, also known to be a substrate for protein kinase C in many cell types (25, 29, 30). This finding is surprising, since the PDGF-induced increase in accumulation of inositol phosphates suggests that turnover of phosphoinositides is stimulated which should lead to production of diacylglycerol, an activator of protein kinase C (28). However, Thompson et al. (32) have recently demonstrated that EGFstrongly stimulates turnover of phosphoinositides in A431 cells without apparent activation of protein kinase C. It appears that the precise relationship between diacylglycerolproduction and activation of protein kinase C is not clear. These data indicate that activation of protein kinase Cis not a prerequisitefor PDGFdependent mitogenesis, corroborating previous work examining the effects of PDGF on protein kinase C-depleted cells (33). Moreover, PDGF treatment appears to result in activation of kinases other thanprotein kinase C which are able to phosphorylate the EGFreceptor. The effects of PDGF and TPA on EGF-dependent, tyrosine-specific autophosphorylation of the EGF receptor were compared in FS4 cells. As previously reported for other cell types, TPA treatmentof FS4 cells blocks the EGF-stimulated increase in EGF receptor phosphotyrosine content. PDGF treatment, however, had no effect. These results differ from those reported for regulation of autophosphorylation of the EGF receptor in WI-38 lung fibroblasts, inwhich PDGF and TPA both inhibit EGF-dependent tyrosine phosphorylation of the EGFreceptor (16,22). The data presented demonstrate that regulation of EGF receptor metabolism by PDGF in foreskin fibroblasts differs from that previously reported for WI-38 lung fibroblasts (16, 17). These differences may be cell type specific, since it has been shown that human lung fibroblast isolates (including WI-38 cells) are refractory to the mitogenic stimulation by EGF, whereas EGF is an effective mitogen for human skin fibroblasts (34, 35). The EGF receptor may be governed by different regulatory systems in thesecell types. In thisregard, two distinct PDGF receptor types have been reportedin human fibroblasts(36), and such receptor heterogeneity could underlie the differences in response to PDGF between FS4 foreskin fibroblasts and W1-38 lung fibroblasts. Acknowledgments-Special thanks toThomas Deuel for providing purified PDGF, to JanVilcek and Pravin Sehgal for the FS4 cells, to James Wang for 80,000 protein antiserum, to Dr. Antony Rosen for protein kinase C, and to JohnMendelsohn for antibody 528. REFERENCES 1. Carpenter, G. (1987) Annu. Reu. Biochem. 56,881-914 2. Ross, R., Raines, E. W., and Bowen-Pope, D. F. (1986) Cell 4 6 , 155-169

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