no. 268, of 5317-5322,1993 vol. 5, pp. 7, of chemistry u ... · pdf filenucleus (22, 23, 28)....

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 7, Issue of March 5, pp. 5317-5322,1993 Printed in U. S. A.

Suppression of Platelet-derived Growth Factor Receptor Tyrosine Kinase Activity by Unsaturated Fatty Acids*

(Received for publication, October 5, 1992)

Lubomir TomGkaS and Ross J. Resnicks From the Section of Biochemistv, Molecular and Cell Biology, Cornell University, Zthma, New York 14853

Treatment of cells with platelet-derived growth fac- tor (PDGF) was previously shown to increase the re- lease of unesterified fatty acids from phospholipids. In view of these observations we examined the effect of various fatty acids on PDGF receptor activity. Unsat- urated (oleic, linoleic, linolenic, and arachidonic) but not saturated (stearic and arachidic) fatty acids signif- icantly inhibited the tyrosine kinase activity of the PDGF receptor in intact cells and membrane prepara- tions. Half-maximal inhibition (I&,) was observed be- tween 60 and 100 p~ and maximal inhibition between 170 and 200 MM. Lysophospholipids and phospholipids had no effect on receptor activity. Activation of endog- enous phospholipase Az by mellitin in vivo or hydrol- ysis of phosphatidylcholine by purified phospholipase A2 in vitro inhibited PDGF receptor autophosphory- lation similar to that of purified fatty acids. Dimeri- zation of PDGF receptors in vivo was significantly reduced by concentrations of arachidonic acid inhibi- tory for receptor kinase activity while the binding of PDGF was only marginally affected. These data sug- gest a possible feedback mechanism resulting in the reduction of PDGF receptor activity by unsaturated fatty acids generated downstream within the PDGF- dependent signal transduction pathway and this effect may be as a direct result of decreased receptor dimer- ization and/or kinase activity.

Platelet-derived growth factor (PDGF)’ receptor belongs to the large family of membrane-associated receptor tyrosine kinases which exhibit similar characteristics upon activation by their respective growth factors. Upon ligand binding recep- tors oligomerize, become phosphorylated on multiple tyrosine residues, alter their cytoplasmic conformations, and increase their ability to phosphorylate tyrosines of other proteins (1- 5). Phosphorylated PDGF @-receptors form complexes with an array of proteins including phospholipase C y 1 (6-9), GTPase-activating protein (GAP) (10-12), phosphatidylino- sitol 3-kinase (13, 14), Src, Fyn, Yes (15), tensin (16), and

* This investigation was supported by U. S. Public Health Service Grant CA-08964 awarded by the National Cancer Institute, Depart- ment of Health and Human Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Department of Genetics, Comenius University, Mlynska dolina B-1, Bratislava, Czechoslovakia.

B To whom correspondence should be addressed. The abbreviations used are: PDGF, platelet-derived growth factor;

EGF, epidermal growth factor; BS3, bis(sulfosuccinimidy1)suberate; GAP, GTPase-activating protein; PMSF, phenylmethylsulfonyl flu- oride; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate- buffered saline; PAGE, polyacrylamide gel electrophoresis; BHK, baby hamster kidney.

Raf-1 serine/threonine kinase (17, 18). Complex formations generally require the association between specific phospho- tyrosines on the PDGF receptor and domains on interacting proteins which are homologous to noncatalytic regions of the src tyrosine kinase (SH2 and SH3) (19-27). Point mutations of these tyrosines abrogate receptor-associated enzyme ac- tivities suggesting the critical role of PDGF receptor auto- phosphorylation in transducing signals from membrane to nucleus (22, 23, 28).

Following PDGF receptor activation multiple events down- stream are enhanced including expression of the proto-onco- genes c-myc and c-fos (29, 30), ion fluxes (31), membrane ruffling (32), and release of unsaturated acids from membrane phospholipids (33-36). Elevation in the level of free fatty acids, specifically arachidonic acid and its metabolites, rep- resents one of the major physiological events in response to numerous mitogenic stimuli (37-40). Two predominant path- ways for their release have been identified. The first requires the direct action of phospholipase Az and the second involves the sequential activity of phosphatidylinositol-specific phos- pholipase C and a diacylglycerol lipase. Phospholipase AQ is responsible for the majority of free fatty acids released, whereas the phospholipase C pathway serves primarily to generate two additional key second messengers, diacylglycerol and inositol trisphosphate, which activate protein kinase C and Ca2’ release, respectively (41, 42).

Two distinct pathways for the regulation of phospholipase A, activity have been elucidated. One requires the activation of a guanine nucleotide-binding protein (43-46) in response to a variety of peptide hormones such as bradykinin, vaso- pressin, angiotensin, bombesin, and thrombin, while the other functions through a growth factor receptor tyrosine kinase like PDGF or EGF (33-35,47-49). Although the exact mech- anisms of phospholipase A2 activation by growth factor recep- tor tyrosine kinases remain unclear, recent studies demon- strated elevated levels of phosphoserine in cytosolic phospho- lipase A, after treatment with EGF and PDGF, respectively, suggesting the involvement of a serine/threonine kinase within these signal transduction pathways (50).

Arachidonic acid, a polyunsaturated fatty acid (5,8,11,14- cis-eicosatetraenoic acid) plays a major role in signal trans- duction. It can be converted to biologically active metabolites such as prostaglandins and thromboxanes by cyclooxygenase, to leukotrienes and lipoxins by lipoxygenase, and to epoxyei- cosatrienoic acid by the monooxygenase activity of cyto- chrome P-450, all of which influence a wide spectrum of physiological events (40). More directly, arachidonic as well as other unsaturated fatty acids can affect the function of biological membranes by their interaction with receptors, transporters, and enzymes associated with the membrane including those with kinase activity.

This paper describes the effect of phospholipids and fatty acids on PDGF receptor tyrosine kinase activity. Specifically,

5317

5318 Suppression of PDGF Receptor by Unsaturated Fatty Acids

unsaturated fatty acids were inhibitory both in vitro and in vivo. Whereas the binding of PDGF to the receptor was not significantly altered, dimerization of the PDGF receptor in intact cells in the presence of arachidonic acid was dramati- cally reduced. We propose that generation of unsaturated fatty acids in response to PDGF may represent an additional mechanism for modulating events dependent upon PDGF receptor kinase activity.

EXPERIMENTAL PROCEDURES

Cell Lines-The cell line BHK C16 overexpressing @subunit of the human PDGF receptor was a generous gift of Dr. A. Kazlauskas (National Jewish Center for Immunology and Respiratory Medicine, Denver). NIH 3T3 fibroblasts were obtained from Dr. R. Bassin (NIH).

Materials-Human recombinant PDGF-BB was purchased from Gibco Bethesda Research Laboratories, [ Y - ~ ~ P ] A T P and Iz5I-PDGF- BB were obtained from Du Pont-New England Nuclear, Iz5I-Protein- A from ICN, anti-phosphotyrosine antibody and purified p21"GAP, Type I1 (51), were generous gifts of Dr. F. McCormick (Chiron). Rabbit anti-human PDGF type A/type B receptor polyclonal anti- body was obtained from UBI, wheat germ lectin-Sepharose 6MB was purchased from Pharmacia LKB Biochemicals, bis(su1fosuccini- midy1)suberate (BS3) from Pierce, aprotinin and sodium suramin from FBA Pharmaceuticals, leupeptin from Chemicon, and sodium orthovanadate was from Fisher. PMSF, N-acetylglucosamine, fatty acids, phosphatidylcholine, mellitin, phospholipase A2, phenylarsine oxide, and rabbit anti-mouse IgG antibody were obtained from Sigma, and all other phospholipids were purchased from Avanti.

Tissue Culture-Cell cultures were maintained in Dulbecco's mod- ified Eagle's medium (DMEM with 4.5 g/liter glucose; Gibco) supple- mented with 10 mM HEPES, pH 7.4, gentamycin (40 pg/ml, Gibco), and 10% newborn calf serum (Gibco), designated as complete me- dium. For experiments, cells were seeded onto appropriate size culture dishes (Falcon) at a density 1-1.5 X lo4 cells per cm2 of complete medium and grown to confluency (3-4 days) in a humidified atmos- phere of 5% Con, 95% air a t 37 "C.

Preparation of Membranes from BHK C16 Cells-Membranes were prepared essentially as described by Williams et al. (52). Confluent cells were scraped, washed three times with ice-cold 50 mM borate, pH 7.4, 150 mM NaCl, and homogenized in 5 volumes of lysis buffer (20 mM borate, pH 10.2, 2 mM EDTA, 2 mM PMSF, 20 pg/ml aprotinin, 10 pg/ml leupeptin, 1 mM sodium suramin) using glass/ glass homogenizer (20 strokes). Suramin was included in the lysis buffer to remove endogenous PDGF associated with the receptor. The homogenate was centrifuged at 30,000 X g for 20 min at 4 "C and the pellet resuspended in buffer A (137 mM NaCI, 3 mM KCl, 4 mM Na2HP04, 1.5 mM KHzP04, pH 7.4,2 mM EDTA, 1 mM dithiothreitol, 10 pg/ml leupeptin, 20 pg/ml aprotinin, 2 mM PMSF). The suspension was centrifuged at 50,000 X g for 30 min at 4 "C and the pellet was washed five times with buffer A. The homogenate was then centri- fuged at 2,000 X g for 15 min at 4 "C to remove cellular debris and nuclei, and the resulting supernatant was centrifuged at 50,000 X g for 30 min at 4 "C. The final pellet was resuspended in 20 mM HEPES/NaOH, pH 7.4, 250 mM sucrose, aliquoted, and stored at

Protein Determination-Protein concentrations were determined by the method of Bradford (53) using bovine serum albumin as a standard.

Extraction of Membrane Proteins-Membranes were mixed with solubilization buffer to a final concentration of 1% Triton X-100,150 mM NaCl, 2 mM PMSF, 10 pg/ml leupeptin, 20 pg/ml aprotinin, 0.1% bovine serum albumin, vortexed and incubated on ice for 30 min. After centrifugation at 100,000 X g for 45 min at 4 "C the supernatant was aliquoted (at protein concentration, 3 mg/ml) and stored at -70 "C.

In Vitro Kinase Assay-Triton X-100 solubilized membrane pro- teins (10 pg) were assayed for PDGF-dependent kinase activity in a final volume of 50 pl of 20 mM HEPES/NaOH, pH 7.4,3 mM MnC12, 0.1% Triton X-100. PDGF-BB (200 ng/ml) was added for 15 min prior to addition of 20 p M [Y-~'P]ATP (specific activity 10,000-15,000 cpm/pmol). After 10 min incubation on ice, reactions were stopped by addition of 12.5 pl of 5 X Laemmli SDS-PAGE sample buffer, boiled for 5 min, and electrophoresis was carried out using 10% acrylamide gels in the buffer system of Laemmli (54). Gels were

-70 "C.

stained with Coomassie Blue, dried, and exposed to Kodak XAR-5 film at -70 "C.

In Vivo Assay for Autophosphorylation of PDGF Receptor-Con- fluent NIH 3T3 cells grown in 60-mm Petri dishes were starved for 16 h in DMEM supplemented with 0.5% newborn calf serum. Cells were washed two times with PBS and covered with serum-free DMEM. After treatment with different agents, PDGF-BB was added to a final concentration of 50 ng/ml for 5 min at 37 "C. Cells were placed immediately on ice, washed twice with ice-cold PBS, solubi- lized with 0.5 ml of solubilization buffer (1% Triton X-100, 50 mM Tris-HC1, pH 8.0,5 mM EDTA, 2 mM sodium orthovanadate, 100 p~ ZnClz, 150 mM NaCl, 10 pg/ml leupeptin, 20 pg/ml aprotinin, 2 mM PMSF), and transferred to centrifuge tubes. Samples were vortexed and incubated on ice for 30 min followed by centrifugation at 100,000 X g for 30 min at 4 "C. Supernatants were mixed with 20 pl of wheat germ lectin-Sepharose 6MB beads prewashed with solubilization buffer, and incubated with agitation for 2 h at 4 "C. Beads were washed twice with 0.5 mI of 1 M NaCI, 20 mM Tris-HC1, pH 8.0,0.1% Triton X-100, twice with 20 mM Tris-HCI, pH 8.0, 0.1% Triton X- 100, resuspended in 1 X Laemmli SDS-PAGE sample buffer and electrophoresis was carried out using 6% acrylamide gels. Separated proteins were analyzed by immunoblotting as described below.

Immunoblot Analysis-Separated proteins were transferred to ni- trocellulose filters for 2.5 h at 250 mA using a semi-dry electroblotter (ISS-ENPROTECH) as described (55). Filters were incubated for 2 h with blocking solution (20 mM Tris-HCI, pH 7.5,150 mM NaCI, 2% nonfat milk). For anti-phosphotyrosine immunoblots, filters were incubated overnight at 4 "C in blocking solution containing anti- phosphotyrosine monoclonal antibody (2 pg/ml), rinsed five times with washing buffer (20 mM Tris-HCI, pH 7.5, 150 mM NaCl, 0.05% Tween 20), and incubated for 2 h at room temperature in blocking solution containing rabbit anti-mouse IgG (14 pglml). For anti-PDGF receptor immunoblots, filters were incubated overnight at 4 "C in blocking solution containing anti-PDGF receptor polyclonal anti- body. After five washes, immunoblots were developed by incubation in blocking solution containing '251-Protein A (1 pCi/ml, 230 pCi/ pg), washed five times, dried, and exposed to Kodak XAR-5 film at -70 "C.

'"I-PDGF Binding in Vivo-In vivo PDGF binding experiments were performed using monolayer cultures in 24-well cluster trays as previously described (56). Confluent cells were starved for 16 h in DMEM supplemented with 0.5% newborn calf serum. Monolayers were rinsed twice with PBS and refed with serum-free DMEM. After cells were treated with lipids, lZ5I-PDGF (-50,000 cpm/well, 3 ng/ml) was added for 5 min at 37 "C, chilled on ice, and washed five times with PBS containing 0.25% bovine serum albumin. Cells were solu- bilized with 1% Triton X-100 containing 0.1% bovine serum albumin and y-radiation was quantitatedin a Beckman y-counter. Nonspecific lz5I-PDGF binding was determined using a 50-fold excess of unlabeled

lZ5I-PDGF Binding in Vitro-PDGF binding in membranes from BHK C16 cells was performed essentially as described (52). Briefly, 20 pg of membrane protein were incubated in a total volume of 0.6 ml of kinase reaction buffer in the presence or absence of lipids with lz5I-PDGF (-60,000 cpm/2 ng) for 20 min at 4 "C. After incubation, membranes were Centrifuged at 12,000 X g for 5 min at 4 "C, washed five times with 0.5 ml of 10 mM Tris-HC1, pH 7.5, 0.15 NaCI, 5% calf serum, and radioactivity was quantitated. Nonspecific "'1-PDGF binding was determined using a 50-fold excess of unlabeled PDGF- BB.

Chemical Cross-linking of PDGF Receptor Dimers in Vivo-Dimer- ization of PDGF receptors in vivo was performed essentially as described (57). Briefly, confluent cells in 100-mm dishes were starved in DMEM with 0.5% calf serum for 18 h, washed two times with PBS, and incubated in 1 ml of serum-free DMEM in the absence or presence of arachidonic acid for 30 min at 37 "C. After a 5-min treatment with PDGF-BB (50 ng/ml), cells were chilled on ice, washed four times with ice-cold PBS, and incubated in 1 ml of ice- cold PBS containing 25 mM HEPES, pH 7.4, and 1.5 mM BS3 for 8 min at 4 "C. Cells were washed twice with ice-cold PBS and incubated with PBS containing 10 mM ammonium acetate for 2 min at 4 "c. Cell monolayers were washed once with ice-cold PBS, solubilized, and PDGF receptor was partially purified as described for the in vivo kinase assay. Protein was separated in a 4.5% SDS gel, and analyzed by immunoblotting using anti-PDGF receptor antibody as described above.

Preparation of L+ids-All lipids (except for lysophospholipids) were stored in chloroform at -20 "C. Aliquots were dried under a

PDGF-BB.

Suppression of PDGF Receptor by Unsaturated Fatty Acids 5319 stream of nitrogen and washed once with diethyl ether. Suspensions of lipids were sonicated in 20 mM Tris-HC1, pH 8.0, in a water bath sonicator a t room temperature until uniform. Lysophospholipids were directly dissolved in Tris-HCI and sonicated. The pH of all lipids were adjusted with NaOH if necessary.

Reproducibility of Data-Each experiment described in this paper was repeated at least two times all with similar results.

RESULTS

Unsaturated Fatty Acids but Not Saturated Fatty Acids Inhibit PDGF Receptor Autophosphorylcltion in Vitro-Be- cause release of arachidonic acid (C204) as well as other fatty acids are a consequence of an enhanced phospholipase activity initiated by PDGF, we investigated their potential to influ- ence PDGF receptor transmembrane signaling. Detergent extracts of membranes prepared from BHK C16 cells that overexpressed the 8-subunit of human PDGFR (by -10-fold) were utilized for assaying the direct effects of fatty acids on PDGF receptor kinase activity in vitro. As shown in Fig. 1, PDGF receptor routinely appeared as two bands, reflecting either a different pattern of glycosylation or proteolytic deg- radation, both of which reacted strongly with anti-PDGF receptor antibody when analyzed by immunoblotting (data not shown). Exposure of receptor to increasing concentrations of arachidonic acid resulted in the progressive reduction in its autophosphorylation. Maximal inhibition was observed at a concentration of 150 pM with an ICs0 of about 60 pM (Fig. 1, A, lanes C-I, and panel B ) . In contrast, the saturated counter- part of arachidonic acid, arachidic acid (C2OO), had no effect within this concentration range (Fig. 1, A, lanes J-P, and panel B ) .

To examine whether this inhibitory property was a feature common to unsaturated fatty acids or specific for arachidonic acid, a selection of commonly occurring fatty acids were screened for their ability to inhibit PDGF receptor autophos-

A Lane A B C D E F G H I J K L M N 0 P C20:4[kM] - - 40 80 120 160200 240 280 - - - - - - - CZOO[FMJ - - - - - - - - - 40 80 120 160200240280 PDGF - t t + t + + + + t t + t t + t

180 kDa

.- Sol\

01 0 c200

C204

20

Y ’ ’ ’ ’ ~ ’ ~ ’

0 40 80 120 160 200 240 280

Fatty acid [FM]

FIG. 1. Titration of arachidonic and arachidic acids on PDGF receptor autophosphorylation in vitro. A, in vitro kinase assays were carried out as described under “Experimental Procedures” in the absence of free fatty acid (lanes A and B ) , or in the presence of increasing concentrations of either arachidonic (C204, lanes C - I ) or arachidic (C200, lanes J-P) acids. Lane A , minus PDGF; lanes B- P, plus PDGF. Samples were analyzed by 10% SDS-PAGE and 32P incorporation was visualized by autoradiography. B, relative levels of receptor autophosphorylation were quantitated using a Betascope (Betagen).

phorylation (Table I). Whereas saturated fatty acids stearic (C180) and arachidic (C2OO) had no effect up to 280 pM, the unsaturated fatty acids oleic (Cl81), linoleic (C182), and linolenic (C183) inhibited PDGF receptor autophosphoryla- tion 7040% at a concentration above 150 p ~ . As indicated by the ICso values shown in Table I, arachidonic acid was the most potent.

It was of interest to determine whether esterified fatty acids were also potent inhibitors of PDGF receptor activity. Phos- phatidylcholine, phosphatidylserine, phosphatidylethanola- mine, and their lyso-derivatives proved ineffective. Only phos- phatidylinositol and lysophosphatidylinositol suppressed ac- tivity but their effective inhibitory concentrations were approximately three times greater than that observed for the unesterified unsaturated fatty acids (Table I). Myo-inositol, the sugar moiety of phosphatidylinositol and lysophosphati- dylinositol, had no effect at concentrations up to 10 mM (data not shown).

An explanation for the diminution of receptor autophos- phorylation by fatty acids could be the activation of a phos- photyrosine phosphatase present within the receptor prepa- rations. To rule out this possibility, sodium orthovanadate (1 mM) and phenylarsine oxide (20 pM), two potent phosphoty- rosine phosphatase inhibitors, were included in the kinase reaction. Both failed to reverse the effects of arachidonic acid suggesting a mechanism consistent with the direct inhibition of kinase activity rather than activation of a contaminating phosphatase (data not shown).

Effect of Arachidonic Acid on PDGF Receptor Kinase Activ- ity Measured by Phosphorylation of p21”GAP“As an alter- native to assaying the autophosphorylation of the PDGF receptor, we tested the effects of arachidonic acid on the phosphorylation of an exogenous substrate. The GTPase- activating protein (GAP) for p21” was selected because it serves as a substrate for PDGF receptor tyrosine kinase activity in vivo (10, 11). When a baculovirus expressed, trun- cated form of purified GAP was incubated with a membrane preparation of PDGF receptor from BHK C16 cells, phos- phorylation occurred in a PDGF-dependent manner (Fig. 2). Although the stoichiometry of PDGF-dependent GAP phos-

TABLE I Effect of lipids on PDGF receptor autophosphorylation in vitro

Kinase assays in vitro were carried out as described under “Exper- imental Procedures,” in the presence of increasing concentrations (0- 300 PM) of lipids. Phosphate incorporation was quantitated using a Betascope (Betagen). Results are an average of two independent experiments. “No effect,” no inhibition observed dp to 300 pM; IC,,, concentration of lipid required for maximal inhibition.

Lipid IC, Maximal IC, inhibition

P M % Phosphatidylcholine No effect Phosphatidylethanolamine No effect Phosphatidylserine No effect Phosphatidylinositol 290f 0 ND” ND

Lysophosphatidylcholine No effect Lysophosphatidylethanolamine No effect Lysophosphatidylserine No effect Lysophosphatidylinositol 2132 37 ND ND

Stearic acid (180) No effect Oleic acid (181) 105 f 1 170f 20 58-C 2 Linoleic acid (182) 85 f 10 205 f 10 75 +5 Linolenic acid (183) 73 f 24 180f 20 75 f O Arachidic acid (200) No effect Arachidonic acid (204) 56 f 4 175k 18 80 2 0

ND, not determined.

5320 Suppression of PDGF Receptor by Unsaturated Fatty Acids

Lane A B C D C20:4 - - + + PDGF - + - +

GAP - FIG. 2. Arachidonic acid inhibits PDGF-dependent phos-

phorylation of GAP in vitro. In vitro kinase assays were carried out as described under “Experimental Procedures” using 0.5 pg of purified p21”’GAP as substrate for the PDGF receptor in the absence (lanes A and B ) or presence (lanes C and D) of 150 ~ L M arachidonic acid (C204). Lanes A and C, minus PDGF; lanes B and D, plus PDGF. Samples were analyzed by 10% SDS-PAGE and 32P incorporation was visualized by autoradiography.

L a n e A B C D E F G H I J K L M N O P c20:4 . . - - - - + + . . - - - . + +

pc I .. + + - - - . I + + . . . . PDGF - + - + - + - + - + - + - + - + 180 kDa

LysoPC- - - . + + . . - - . - + + . .

” .. ~.

-PLA2 +PLA2

FIG. 3. Reconstitution of phospholipase (PL) Aa-mediated inhibition of PDGF receptor kinase activity in vitro. Lipids (200 p ~ ) were preincubated for 15 min at 30 “C in 20 mM HEPES, pH 7.4, 0.1% Triton X-100 in the absence (lanes A-H) or presence (lanes I-P) of bee venom phospholipase AI (1 unit). I n vitro kinase assays were then carried out as described under “Experimental Pro- cedures.” Lanes A, B, I, and J , control, no lipid; lanes C, D, K, and L, phosphatidylcholine (PC); lanes E, F, M, and N , lysophosphatidyl- choline (Lyso-PC); or lanes G, H, 0, and P, arachidonic acid (C20:4). Lanes A, C, E, G, I , K, M, and 0, minus PDGF; lanes B, D, F, H, J , L, N , and P, plus PDGF. Samples were analyzed by 10% SDS-PAGE and 32P incorporation was visualized by autoradiography.

phorylation was relatively low, addition of 150 p~ arachidonic acid to the kinase reaction inhibited its phosphorylation greater than 90%.

Hydrolysis of Phosphatidylcholine by Bee Venom Phospho- lipase A2 Results in the Inhibition of PDGF Receptor Auto- phosphorylation in Vitro-Phospholipase A2 is considered to be one of the key enzymes in liberating free fatty acids from phospholipids in response to various hormones and growth factors, including PDGF. We therefore tried to mimic this process in vitro by pretreatment of 200 p~ egg yolk phos- phatidylcholine, containing a mixture of unsaturated fatty acids at the sn-2 position, with bee venom phospholipase A2 (Fig. 3). Whereas this concentration of phosphatidylcholine did not affect the PDGF receptor kinase activity (lanes C and D ) as compared with the control (lanes A and B ) , its hydrol- ysis and the subsequent release of unsaturated fatty acids resulted in an inhibition of receptor autophosphorylation (lanes K and L) similar to that observed with 200 p~ arachi- donic acid (lanes G and H). The concomitant generation of an equimolar amount of lysophosphatidylcholine, which has no effect alone (lanes E and F ) did not prevent the inhibition by the released unsaturated fatty acids. Phospholipase A2 alone (lanes I and J), pretreatment of lysophosphatidylcho- line (lanes M and N ) , or arachidonic acid (lanes 0 and P ) with phospholipase A2 did not alter PDGF receptor kinase activity.

Arachidonic Acid Inhibits PDGF Receptor Tyrosine Kinase Activity in Intact Cells-The hydrophobic nature of fatty acids makes it possible to examine their effects in vivo because of their ability to traverse the plasma membrane. To test the effects of fatty acids on PDGF-dependent tyrosine autophos-

phorylation of the PDGF receptor in intact cells, an assay based on precipitation of cellular glycosylated proteins by wheat germ lectin beads followed by anti-phosphotyrosine immunoblot was used. Elevated levels of phosphorylation were detected in a 180-kDa band representing the PDGF receptor after cells were treated with PDGF-BB, whereas no detectable tyrosine phosphorylation was evident without PDGF treatment (Fig. 4A, lanes A and B ) . Increasing concen- trations of arachidonic acid decreased autophosphorylation of the PDGF receptor approximately 80% at a concentration of 120 pM (Fig. 4, A, lunes C-I, and panel B ) . The ICSo for arachidonic acid in vivo was calculated to be about 70 p ~ . In comparison, oleic, linoleic, and linolenic all inhibited PDGF receptor tyrosine kinase activity to a similar extent, whereas stearic and arachidic had no effect (data not shown). Conse- quently these data correlate closely with the i n vitro data presented earlier.

I t was of interest to determine whether the protein tyrosine kinase of a related growth factor receptor might also be sensitive to unsaturated fatty acids. We therefore tested the effect of arachidonic acid on the tyrosine kinase activity of the EGF receptor. After exposure of cells to EGF (250 ng/ml) for 5 min, autophosphorylation of a 170-kDa band represent- ing the EGF receptor was detected. Phosphotyrosine content of the receptor was not affected by pretreatment of cells with 150 p~ arachidonic acid (data not shown) which is in agree- ment with observations made by Casabiell et al. (58).

Mellitin Induces an Inhibition of PDGF Receptor Autophos- phorylation in Vivo-Mellitin, a water-soluble amphipathic polypeptide composed of 26 amino acids which is the active constituent of bee venom (59, 60), was shown to be a potent activator of endogenous phospholipase A2 in cell culture (61). A sublytic concentration of mellitin (10 pg/ml) activated endogenous phospholipase A2 in 3T3-4a mouse fibroblasts to hydrolyze about 30% of its phosphatidylcholine after a 15- min treatment without activating phospholipase C (61). We therefore took advantage of this biological property and tested whether activation of endogenous phospholipase A2 would diminish the PDGF receptor autophosphorylation-like addi-

Lane A B C D E F C H I C20:41uM1 - . 10 25 SO 7s 100 125 150

A

#!= 0 ... , . *\

0 25 50 75 100 125 150

C204 [pM]

FIG. 4. Inhibition of PDGF receptor tyrosine kinase activ- ity in vivo. A, cells were pretreated for 30 min at 37 “C in the absence (lanes A and B ) or presence of increasing concentrations of arachidonic acid (C20:4; lanes C - I ) , and in vivo kinase assay was carried out as described under “Experimental Procedures.” Lane A, minus PDGF; lanes B-I, plus PDGF. Samples were subjected to 6% SDS-PAGE and analyzed for phosphotyrosine content by immuno- blotting with anti-phosphotyrosine antibody and ‘251-Protein A and results were visualized by autoradiography. B, relative levels of phos- photyrosine were determined by cutting corresponding bands from nitrocellulose filter.

Suppression of PDGF Receptor by Unsaturated Fatty Acids 5321

tion of purified fatty acids. After pretreatment of cells with 10 pg/ml of mellitin for 15 min, approximately 80% of the PDGF-stimulated tyrosine activity was lost (Fig. 5). This was not due to a reduction in the number of receptors as confirmed by an anti-PDGF receptor immunoblotting assay (data not shown). When tested in vitro, 10 pg/ml mellitin had no effect on the autophosphorylation of PDGF receptor, excluding its direct inhibition of the kinase (data not shown).

Arachidonic Acid Does Not Affect '251-PDGF Binding to PDGF Receptor-The association of PDGF with its receptor is essential for activity, and any impairment of this interaction would result in a loss of kinase activity. To exclude the possibility that the effect of fatty acids might be due to interference of PDGF binding to its receptor, direct measure- ment of 12'I-PDGF binding was examined. In vitro, '251-PDGF binding to membranes was slightly enhanced (120%) in the presence of 150 ~ L M arachidonic acid when compared with the control. When l2'1-PDGF binding to NIH 3T3 cells was measured, arachidonic acid (120 p ~ ) decreased the specific binding of 12'I-PDGF less than 20% (data not shown). Ac- cording to these data, we conclude that the reduction in PDGF receptor kinase by unsaturated fatty acid activity lies down- stream from growth factor binding to its receptor.

Arachidonic Acid Inhibits the Dimerization of PDGF Recep- tors in Vivo-Following binding of PDGF, the receptor di- merizes, and this process is believed to be essential for its tyrosine kinase activity. The effect of arachidonic acid on PDGF receptor dimerization in vivo was therefore investi- gated. The treatment of cells with PDGF was carried out as described for the in vivo kinase assay, followed by cross- linking of PDGF receptor dimers with BS3 and immunoblot- ting with anti-PDGF receptor antibody. As shown in Fig. 6, an 80% inhibition of dimer formation was observed in the presence of 120 p~ arachidonic acid, whereas the total amount of PDGF receptors remained essentially unchanged. The de- crease in dimerization by this concentration of arachidonic acid was comparable with the reduction in receptor tyrosine kinase activity suggesting that the mechanism of action of arachidonic acid on PDGF receptor might be due to the suppression of dimer formation.

DISCUSSION

Treatment of cells with a variety of hormones and growth factors including PDGF leads to the release of unsaturated fatty acids from the sn-2 position of phospholipids. Prior to being metabolized these lipids can alter a variety of biological events associated with the plasma membrane. These include ion transport (62-64), cyclic nucleotides formation (65-68), the activity of regulatory proteins such as p21'""GAP (69,70), and protein kinase activity (71-73).

The effect of unsaturated fatty acids on protein kinase

Lane A B C D melittin - - + + PDGF - + - +

180 kDa 1 * FIG. 5. Mellitin-mediated inhibition of PDGF receptor au-

tophosphorylation in vivo. Cells were preincubated for 10 min at 37 "C in the absence (lanes A and B ) or presence (lanes C and D) of mellitin (10 pglml). In uioo kinase assay was carried out as described under "Experimental Procedures." Lanes A and C, minus PDGF; lanes B and D, plus PDGF. Samples were subjected to 6% SDS- PAGE and analyzed for phosphotyrosine content by immunoblotting with anti-phosphotyrosine antibody and lZ5I-Protein A and results were visualized by autoradiography.

Lane A B C D C20:4 - - t t PDGF - t - +

360 kDs

FIG. 6. Effect of arachidonic acid on PDGF-stimulated di- merization of PDGF receptor in vivo. Cells were incubated for 30 min at 37 "C in the absence (lanes A and B ) or presence (lanes C and D) of 150 p~ arachidonic acid (C20:4) and chemical cross-linking using BS3 was performed as described under "Experimental Proce- dures." Lanes A and C, minus PDGF; lanes B and D, plus PDGF. Samples were subjected to 4.5% SDS-PAGE, PDGF receptor mono- mers (180 kDa) and dimers (360 kDa) were analyzed by immunoblot- ting with anti-PDGF receptor antibody and 1251-Protein A and results were visualized by autoradiography.

activity depends on characteristics unique to each enzyme. Protein kinase C , a lipid requiring enzyme, was stimulated 3- fold by 200 p~ arachidonic acid (71); CAMP-dependent pro- tein kinase activity was significantly inhibited (73). Our work demonstrates that PDGF receptor tyrosine kinase activity at this concentration was strongly inhibited both in vitro and in vivo (Figs. 1 and 4), but under identical conditions the tyrosine kinase activity of EGF receptor in vivo was not affected (data not shown). This is in agreement with the work of Casabiell et at. (58), who demonstrated that while EGF receptor auto- phosphorylation in vivo was not affected by 100 p~ oleic acid, EGF-induced increases in cytosolic [Ca2+], membrane poten- tial and inositol 1,4,5-trisphosphate were suppressed. Al- though PDGF and EGF receptors are transmembrane recep- tor tyrosine kinases, their molecular organization differs sig- nificantly such that they have been classified into different subclasses (5). EGF receptor is a member of subclass I char- acterized by two cysteine-rich repeat sequences within the extracellular domain. The PDGF receptor belongs to subclass I11 which is characterized by three or five immunoglobulin repeats within the extracellular domain and by the presence of hydrophilic insertion sequences within the tyrosine kinase domain. These structural differences may account for their differential sensitivity to unsaturated fatty acids.

Subclass I11 receptors including PDGF receptor and colony- stimulating factor type 1 undergo dimerization upon ligand binding both in vitro (74, 75) and in vivo (76, 77). In intact cells both noncovalent and covalent disulfide-bridged dimers of receptors have been identified (57, 77). It was proposed that covalent dimerization participates in receptor internali- zation while noncovalent dimerization appears critical for receptor kinase activation. In view of this model we investi- gated whether arachidonic acid might influence kinase activ- ity due to inhibition of dimer formation. For consistency, cells were pretreated with arachidonic acid as described for the in vivo kinase assay although these conditions yielded a subop- timal amount of dimers as a consequence of rapid receptor internalization and low cross-linking efficiency at elevated temperature (57). Treatment of cells with arachidonic acid inhibited dimerization 80% which correlated closely with the suppression of kinase activity. This result implies that dimer-

5322 Suppression of PDGF Receptor by Unsaturated Fatty Acids

ization of PDGF receptor plays a critical role in its activation, although it does not exclude the possibility that dimerization is a consequence of receptor autophosphorylation. We propose that unsaturated fatty acids bind to domain(s) on the PDGF receptor responsible for either dimerization or catalytic activ- ity. Our data do not distinguish between either of these possibilities, but a reduction in either would result in the suppression of receptor-mediated signal transduction.

PDGF receptor forms an association with GAP in response to PDGF which requires the phosphorylation of a tyrosine residue within the kinase insert of the PDGF receptor. (19, 20, 22). This interaction leads to the subsequent phosphoryl- ation of GAP by the receptor. We have demonstrated that the phosphorylation of GAP by the PDGF receptor in uitro is susceptible to inhibition by arachidonic acid (Fig. 2). GAP was also shown to physically interact with arachidonic acid (78, 79) retarding its ability to stimulate the GTPase activity of ~ 2 1 " ~ in uitro. In uiuo, ~ 2 1 ' " ~ accumulates in its active GTP- bound form after PDGF treatment (80, 81). Together these data suggest that arachidonic acid may play a dual role within the PDGF-dependent signal transduction pathway. Rapid elevations in the intracellular concentration of arachidonic acid in response to PDGF (33-36) would prevent the consti- tutive activation of this pathway by inhibition of the receptor tyrosine kinase. In conjunction with internalization, this may contribute to the down-regulation of the receptor. Conversely, the inability of GAP to potentiate the GTPase activity of p21'" in the presence of arachidonic acid would preserve the activity of downstream events.

In normal cells, mitogenesis reflects a delicate balance between positive and negative control mechanisms. We pro- pose that part of this regulatory network for PDGF receptor involves the level of unesterified unsaturated fatty acids mod- ulated by the metabolic state of the cell.

Acknowledgments-We thank Drs. Richard Cerione, Leon Heppel, and David Shalloway for comments and critical reading of the man- uscript. This work was completed in the laboratory of the late Efraim Racker to whom we were privileged to be both students and friends.

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5. 4.

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10.

11.

12.

13.

14.

15.

16.

17.

18.

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21.

REFERENCES

Yarden. Y.. and Ullrich. A. (1988) Eiochernistrv 27.3113-3119 Hunter, T., and Cooper, J. A. (1985) Annu. Reu. Eiochern. 64,897-930

Carpenter,'G. (1987) Annu. keu. Eiochern. 66,-881-914 Williams, L. T. (1989) Science 2 4 3 , 1564-1570 Ullrich, A., and Schlessinger, J. (1990) Cell 6 1 , 203-212 Kumjian, D. A., Wahl, M. I., Rhee, S. G., and Daniel, T. 0. (1989) Proc.

Meisenhelder, J., Suh, P.-G., Rhee, S. G., and Hunter, T. (1989) Cell 6 7 , Natl. Acad. Sci. U. S. A. 86,8232-8236

Morrison, D. K., Kaplan, D. R., Escobedo, J. A., Rapp, U. R., Roberts, T.

Wahl. M. 1.. Olashaw. N. E.. Nishibe. S.. Rhee. S. G.. Pleeer. W. J.. and

1109-1122

M., and Williams, L. T. (1990) Mol. Cell. Eiol. 10,2359-2366

Ca&iiti;, G. (1989) M O L Cell. E ~ o L ' ~ , 2934-2943

L. T. (1990) Cell 61,125-133

I~~ ~~ I~ ~ I

Kaplan, D. R., Morrison, D. K., Wong, G., McCormick, F., and Williams,

Kazlauskas, A., Ellis, C., Pawson, T., and Cooper. J. A. (1990) Science 247. 157R-15R1

Molloy, C. J., Bottaro, D. P., Flemming, M. S., Gibbs, J. B., and Aaronson,

Coughlin, S. R., Escobedo, J. A., and Williams, L. T. (1989) Science 2 4 3 ,

"._ "" S. A. (1989) Nature 342 , 711-714

1191-1194 Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A,,

Kypta, R. M., Goldberg, Y., Ulug, E. T., and Courtneige, S. A. (1990) Cell

Davis, S., Lu, M. L., Lo, S. H., Lin, S., Butler, J. A,, Druker, B. J., Roberts,

Morrison. D. K., Kaolan. D. R.. R ~ D u , U., and Roberts, T. M. (1988) Proc.

.~ .~~

Kapeller, R., and Soltoff, S. (1991) Cell 6 4 , 281-302

62,481-492

T. M., An, Q., and Chen, L. B. (1991) Science 262,712-715

Natl. A'cad. Sei. l.? S. A. 86,8855-8859

M.. and Williams. L. T. (1989) Cell 68.649-657 Morrison D. K., Kaplan, D. R., Escobedo, J. A., Rapp, U. R., Roberts, T.

Kazlauskas, A,, and'cooper, J. A. (1989) Cell 68,1121-1133 Kazlauskas, A,, Durden, D. L., and Cooper, J. A. (1991) Cell Regul. 2,413-

Escobedo, J. A,, Kaplan, D. R., Kavanaugh, W. M., Turck, C. W., and 425

Williams, L. T. (1991) Mol. Cell. Eiol. 1 1 , 1125-1132

22. Kashishian, A., Kazlauskas, A., and Cooper, J. A. (1992) EMEO J. 11 , 127R-12R2

23.

24.

25.

26. 27.

28.

29.

Fantl, W. J., Escobedo, J. A., Martin, G . A., Turck, C. W., del Rosario, M., Kli pel, A , Escobedo, J. A., Fantl, W. J., and Williams, L. T. (1992) Mol.

McGlade, C. J., Ellis, C., Reedijk, M., Anderson, D., Mbamalu, G., Reith, A. D., Panayotou, G., End, P., Bernstein, A., Kazlauskas, A., Waterfield, M. D., and Pawson, T. (1992) MOL Cell. Ewl. 12,991-997

- - . - - - "

McCormlck, F., and Williams, L. T. (1992) Cell 69,413-423

&U. EWl. 12,1451-1459

Pawson, T. (1988) Onco e m 3,491-495 Koch, C. A., Anderson 6., Moran, M. F., Ellis, C., and Pawson, T. (1991)

Yu, J.-C., Heidaran, M. A., Pierce, J. H., Gutkind, J. S., Lombardi, D. Science 262,668-674

Cochran, B. H., Zullo, J., Verma, I. M., and Stiles, C. D. (1984) Science Ruggiero, M., and Aaronson, S. A. (1991) Mol. Cell. EioL 11,3780-37&

226.1080-1082 30. Muller, R., Bravo, R., Burckhardt, J., and Curran, T. (1984) Nature 312 ,

31. Lo ez Rivas, A., Stroohant, P., Waterfield, M. D., and Rozengurt, E. (1984)

32. Mellstrom, K., Heldin, C.-H., and Westermark, B. (1988) Exp. Cell Res.

I~ ~~ ~~ ~

716-720

EMBO J. 3,939-944

177. RA7-.759 33.

34.

36. 35.

37.

39. 38.

Levine ".

Van den Bosch,.H. (1980) Eiochirn. Biophys. Acta 6 0 4 , 191-246

Chang, J., Musser, J. H., and McGregor, H. (1987) Eiochern. Pharrnacol. Irvine, R. F. (1982) Eiochern. J. 204,3-I6

36.2429-2436 40. Shimizu, T., and Wolfe, L. S. (1990) J. Neurochern. 66,1-15 41. Nishizuka, Y. (1984) Nature 308,693-698 42. Berridge, M. J. (1990) J. Eiol. Chern. 266,9583-9586 43. Ax:?$:_J.,.Fwch, R. M., and Jelsema, C. L. (1988) Trends Neurol. Sci.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53. 54. 55. 56.

58. 57.

59.

60.

62. 61.

63.

64.

66. 65.

67.

68. 69.

70.

71.

72. 73.

74.

75.

76.

77. 78.

79.

80.

81.

Murayama, T., Kajiyama, Y., and Nomura, Y. (1990) J. Biol. Chern. 2 6 6 , 11, 11 I-LIJ

Burch, R. M., and Axelrod, J. (1987) Proc. Natl. Acad. Sci. U. S.

Gu ta, K., Diez, E., Heasley, L. E., Osawa, S., and Johnson, G. L.

Gronich, J. H., Bonventre, J. V., and Nemenoff, R. A. (1988) J. Eiol.

Bonventre, J. V., Gronich, J. H., and Nemenoff, R. A. (1990) J. Eiol.

4290-4295

6374-6378

&erne 249,662-666

263,16645-16651

265.49.74-4938

A. 8 4 ,

(1990)

Chern.

Chem.

Goldberg, H. J., Viegas M. M., Margolis, B. L. Schlessinger, J., and

Lin, L.-L., Lin, A. Y., and Knopf, J. L. (1992) Proc. Natl. Acad. Sci. U. S. A.

Halenbeck, R., Crosier, W. J., Clark, R., McCormick, F., and Koths, K.

Williams, L. T., Tremble, P. M., Lavin, M. F., and Sunday, M. E. (1984)

Bradford, M. M. (1976) Anal. Eiochern. 72,248-254 Laemmll, U. K. (1970) Nature 277,680-685 Kyhse-Andersen, J. (1984) J. Eiophys. Eiochern. Methods 10,203-209 Bowen-Po e D F and Ross, R. (1985) Methods Enmmol. 109,69-100 Li, W., a n i ichies'iin er J (1991) Mol. Cell. Biol. 11,3756-3761 Casabiell, X., Pandielya, k.; and Casanueva, F. F. (1991) Eiochern. J. 2 7 8 ,

Scroeder, E., Lubke, K., Lehman, M., and Beetz, I. (1971) Experientia

Habermann, E. (1972) Science 177,314-322

Kim, D., and Clapham, D. E. (1989) Science 244,1174-1176 Shier, W. T. (1979) Proc. Natl. Acad. Sei. U. S. A. 76,195-199

Ordway, R. W.. Walsh. J. V., Jr., and Singer. J. J. (1989) Science 244.

---. ~ - - ~ - ~ ~ -

Skorecki, K. L. (1990)'Eiochern. J. 267,461-465'

89,6147-6151

(1990) J. Eiol. Chern. 266,21922-21928

J. Ewl. Chern. 269,5287-5294

679-687

(Easel) 1 6 , 764-765

1176-1179 - - . - - - . - Lambert, I. H. (1991) Cell. Ph si01 Eiochern. 1, 177-194 Fain J. N., and She herd R k. (i975) J. Eiol. Chern. 260,6586-6592 Maliieri, J. A., ShepRerd, k. E. , and Fain, J. N. (1975) J. Etol. Chern. 2 6 0 , 659.7-fi59R

Orly, J., and Schramm, M. (1975) Proc. Natl. Acad. Sci. U. S. A. 72,3433-

Tsai. M.-H., Yu, C.-L., Wei, F.-S., and Stacey, D. W. (1989) Scrence 2 4 3 , Wallach, D., and Pastan, 1. (1976) J. Eiol. Chern. 261,5802-5809

_I"_ ""_ 3437

Golubic, M., Tanaka, K., Dobrowolski, S., Wood, D., Tsai, M.-H., Marshall,

McPhail, L. C., Clayton, C. 6:. and 'Sayderman, R. (1984) Science 224 , M., Tamanoi, F., and Stace D W (1991) E M E O J . 10, 2897-2903

522-526

699-695 Murakami K., and Routtenberg A. (1985) FEBS Lett. 192 , 189-193 Speizer, L.' A., Watson, M. J., ind Brunton, L. L. (1991) Am. J. Physiol.

"I

261.109-114

Seifert, R. A,, Hart, C. E., PI , J.. and Bowen-P,

. ~~.~ ., ~

1, J., Lautwein, A., Frech. M A 4 E O J. 10,1325-. ~ ~.

Pric. datl. Acad. Sci. U. S. A. 87,. 5993-5997

U. S. (1990) J. Bcol. Chem. 265,20437-20442

Satoh T. Endo, M., Nakafuku, M., Nakamura, S., and Kaziro, Y. (1990)

Gibbs, J. B., Marshall, M. S., Scolnlck, E. M., Dixon, R. A. F., and Vogel,