MOLECULAR AND CELLULAR BIOLOGY, May 1991, p. 2785-2793 0270-7306/91/052785-09$02.00/0 Copyright C) 1991, American Society for Microbiology

Vol. 11, No. 5

Ras GTPase-Activating Protein Physically Associates with Mitogenically Active Phospholipids MEN-HWEI TSAI,'* MARGARET ROUDEBUSH,1 STEVEN DOBROWOLSKI,l CHUN-LI YU,' JACKSON B. GIBBS,2 AND DENNIS W. STACEY' Department of Molecular Biology, The Cleveland Clinic Foundation, Cleveland, Ohio 44106,' and Department of Molecular Biology, Merck Sharp & Dohme Research Laboratory, West Point, Pennsylvania 194862 Received 13 February 1990/Accepted 26 February 1991

The physical interaction between GTPase-activating protein (GAP) and lipids has been characterized by two separate analyses. First, bacterially synthesized GAP molecules were found to associate with detergent-mixed micelles containing arachidonic but not with those containing arachidic acid. This association was detected by a faster elution time during molecular exclusion chromatography. Second, GAP molecules within a crude cellular lysate were specifically retained by a column on which certain lipids had been immobilized. The lipids able to retain GAP on such columns were identical to those which were shown previously to be most active in blocking GAP activity. The association between lipids and GAP was dependent upon magnesium ions. Lipids unable to inhibit GAP activity were also unable to physically associate with GAP. The tight association of GAP with these lipids was predicted by and helps to rationalize their ability to inhibit GAP activity.

Cellular Ras proteins play an important role in controlling cellular proliferation by acting as a component of the proliferative signal transduction pathway (2, 20). Recently, a cytoplasmic protein was identified which stimulates more than 100-fold the GTPase activity of normal Ras but not that of its oncogenic mutant (27). Since the GTPase-activating protein (GAP) can convert biologically active Ras-GTP into the inactive Ras-GDP complex, GAP may be a negative regulator of Ras protein (34). On the other hand, other analyses indicate that GAP might be a Ras effector protein (1, 5, 24). In either case, both GAP activity and Ras activity are likely to be critical in the control of cellular proliferation. On the basis of microinjection studies, we previously reported that the biological activity of Ras might be controlled by phospholipids (32). Consistent with this hypothesis, GAP activity (and hence the nucleotide status of Ras) was found to be inhibited by certain lipids (30). Lipids whose metabolism is altered during mitogenic stimulation (e.g., phosphatidic acid [PA], phosphatidylinositol phosphates, and arachidonic acid) were most active in blocking GAP activity, while more abundant lipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) were totally inactive. Furthermore, in studies of mitogen-stimulated NIH 3T3 cells, a lipid was identified which had the ability to inhibit GAP activity. This lipid was produced within 3 min of mitogen stimulation but only in subconfluent cells (33). These biochemical studies, therefore, support the suggestion that GAP might be inhibited by certain lipids. The production of lipids able to inhibit GAP activity might represent a novel mechanism for regulating the activity not only of Ras but also of ras-related genes. To test this possibility, the R-ras and Rho proteins (which have considerable biochemical and sequence homology with Ras) were analyzed. As with Ras, it was found that each of these related proteins failed to be stimulated by their respective GAP in the presence of lipids which are similar but not identical to those able to inhibit Ras GAP (28). *

The physical mechanism responsible for this biochemical interaction between lipid, GAP, and Ras protein is unclear. Since Ras must be associated with the plasma membrane to be biologically active, GAP must move to the membrane to interact with Ras. In addition, recent studies indicate that at least a portion of cellular GAP molecules become associated with activated growth factor receptor molecules, suggesting that some GAP molecules may translocate to the membrane in response to mitogenic stimulation (6, 13, 19). The placement of GAP at or near the membrane makes it possible that GAP directly interacts with membrane lipid, as would be required for these lipids to regulate GAP activity. In any case, if lipids play a physiological role in the control of GAP activity it is likely that they have the ability to physically associate with GAP molecules. These studies were designed to directly investigate the possible physical association between lipids and GAP. Two independent types of analyses are employed for this demonstration. The phospholipids shown here to associate with GAP were identical to those shown previously to be most active in blocking GAP activity. MATERIALS AND METHODS

Preparation of phospholipid liposome. Phospholipids (0.5 to 1.0 mg) (Sigma) and arachidonic acid (0.5 mg) (Sigma) were dissolved in chloroform and dried under vacuum in a glass tube (12 by 75 cm). The thin layer of lipid coating the glass was suspended in 1 ml of 0.1 M Tris-HCI, pH 7.5, and sonicated in an ice bath for 30 s by inserting a titanium

microtip (Fisher, model 300 Sonic Dismembrator) into the bottom of the glass tube. Half-maximum power output was used for liposome-micelle preparation. Preparation of mouse brain cell extract. Cytoplasmic extract containing GAP activity was prepared from mouse brain. The cerebra from ten animals were homogenized in ice-cold hypotonic buffer (10 mM Tris-HCI [pH 7.5], 5 mM MgCl2, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 3,000 x g for 10 min to remove the unbroken cells. The resulting supernatant was then centrifuged at 100,000 x g for 30 min to

Corresponding author. 2785

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TSAI ET AL.

obtain a clarified supernatant fraction. The cytoplasmic extract containing GAP protein activity (5 mg/ml) was stored at -80°C until used (27, 30). Removal of GAP activity by sedimentation with phospholipid liposome. One milliliter of mouse brain extract was incubated with 200 ,ul of phospholipid liposome (1 mg/ml) in an Eppendorf tube at 30°C for 30 min. The phospholipids in the form of liposomes were then pelleted by centrifugation with an Eppendorf 5415 centrifuge at the maximum speed for 20 min. The GAP activity remaining in the supernatant was then tested for its ability to stimulate Ras GTPase activity as described below. Incubations with purified bovine brain GAP were carried out in 20 mM Tris-HCl (pH 7.5)-3 mM MgCI2-2 mM dithiothreitol-150 mM NaCl with 5 mg of bovine serum albumin (Sigma) per ml added. Immunoprecipitation of Ras by monoclonal antibody Y13259. Nucleotide-free Ha-Ras (2 ,uM) was incubated for 20 min at 30°C with 1 ,uM [a-32P]GTP (3,000 Ci/mmol; Amersham) in 50 ,ul of Tris-HCl buffer, pH 7.5, containing 2 mM dithiothreitol without added MgC12. GTPase reaction was initiated by the addition of MgCI2 (with or without supernatants prepared as described above) in 150 RIl of reaction buffer (final concentrations, 20 mM Tris-HCl [pH 7.5], 3 mM MgC12, 0.15 M NaCl, 1 mM dithiothreitol). After incubation at 30°C for one h, Ras was immunoprecipitated by monoclonal antibody Y13-259 and protein A-Sepharose beads coated with rabbit antibody to rat immunoglobulin G. Bound nucleotides were released from the immunoprecipitate by boiling for 3 min. Bound nucleotides were resolved on polyethyleneimine cellulose thin-layer chromatography plates (EM Science) in 1 M potassium phosphate, pH 3.4, and visualized by autoradiography. Ha-Ras protein was prepared in a bacteria expression system as previously described (30). Preparation of PA AFFI-GEL 102 affinity column for GAP. Arachidic acid was conjugated chemically to the amino group of AFFI-GEL 102 (Bio-Rad) by mixing the following three solutions in an end-over-end mixer overnight at room temperature. Solution 1 (gel suspension): 10 ml of AFFIGEL 102 was washed three times with 100 ml of acetate buffer (pH 4.5). Solution 2 (carbodiimide solution): 150 mg of carbodiimide was dissolved in 15 ml of acetate buffer (pH 4.5). Solution 3 (ligand [arachidic acid] solution): the excess amount of arachidic acid (200 mg) (Sigma) was dissolved in 5 ml of chloroform. This 5 ml of chloroform solution was divided into 10 glass test tubes (12 by 75 cm). A total of 2 ml of acetate buffer (pH 4.5) was added to each tube after the chloroform was dried. The dried lipid was suspended in the buffer by sonication as described above. About 2 ml of arachidic acid-conjugated AFFI-GEL 102 was packed on a column. The column was then extensively washed with Tris-HCl buffer (pH 7.5) containing 5 mM MgCl2-50 mM NaCl. PA (arachidonyl, stearoyl), arachidonic acid, or other phospholipid liposomes were then slowly passed through the column. The lipids were retained by the conjugated AFFIGEL, presumably by hydrophobic interaction with the conjugated arachidic acid since unconjugated AFFI-GEL did not retain the lipids. Elution of GAP from PA affinity column. Five hundred microliters of mouse brain extract was then applied to the PA (13-arachidonyl, -y-stearoyl)-immobilized AFFI-GEL 102 column and washed with 20 ml (10 column volumes) of washing buffer (20 mM Tris, 5 mM MgCl2, 50 mM NaCl [pH 7.5]). After the washing, GAP was eluted with 20 mM Tris buffer containing 10 mM EDTA. GAP was detected by Western blotting (immunoblotting) with anti-GAP antibody

MOL. CELL. BIOL.

no. 638. GAP activity was tested by filter binding assay as described above (except that 15 mM MgCl2 was added in the reaction mixture). Western blotting was performed as follows. Western blotting. Proteins were first resolved on a sodium dodecyl sulfate (SDS)-10% polyacrylamide gel, which was then rinsed in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol) for 15 min. Proteins were transferred to nitrocellulose (0.45 ,uM; Schleicher & Schuell) by electrophoresis at 500 mA for 1 h. The nitrocellulose blot was incubated in 5% nonfat dry milk-phosphate-buffered saline (pH 7.5) for 1 h at room temperature. GAP was then immunostained with rabbit antisera against the amino or carboxyl terminus of GAP. GAP was visualized by incubating it with 4-chloro-naphthol and H202. Whereas the determinations reported here were performed with carboxylterminal antipeptide antibodies, most of these analyses were also performed with amino-terminal antibodies. Size exclusion chromatography in the presence of mixed micelles. Sepharose 4B-200 size exclusion resin in a column (1 by 40 cm) was equilibrated with Tris-HCl buffer (20 mM Tris-HCl [pH.7.5], 5 mM MgCl2, 2 mM dithiothreitol, 0.15 M NaCl, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride). GAP was prepared from a bacterial expression system and purified through two chromatographic steps (7). Briefly, the bacterial cells were lysed with a French pressure cell in a solution containing a variety of protease inhibitors. Treatment with DNase preceded centrifugation and chromatography on a DEAE-Sephacel column (Pharmacia-LKB). The active fractions were concentrated and applied to a Sephadex G-100 size exclusion column. Active fractions were concentrated and utilized. The partially purified GAP was incubated in the micelle solution (see below) for 30 min at 30°C prior to passage over the column. The same column was used for chromatography of samples in arachidic and arachidonic acid, with extensive washing between runs. In several repeat analyses, the order of chromatography of these two samples was altered without changing the results. The arachidonic acid or arachidic acid (300 ,ug) was suspended in aqueous solution as described above. Filter binding assay. Purified bacterial synthesized Ras was first preloaded with [y-32P]GTP by incubating it in 20 ,ul of incubation buffer (20 mM Tris-HCl [pH 7.5], 1 mM dithiothreitol, and 10 ,uM [_y-32P]GTP [10 Ci/mmol; Amersham]) at 30°C for 10 min. GTPase activity was then assayed by incubating the [_y-32P]GTP-loaded Ras at 30°C in 30 ,ul of reaction buffer (20 mM Tris-HCl [pH 7.5], 1 mM dithiothreitol, 1 mM MgCl2, 3 mM unlabeled GTP). After a 30 min incubation, the reaction mixture (0.45 ,uM) was filtered on nitrocellulose filters (pore size, 25 ,um; GA 85, Schleicher & Schuell) and washed at once with 10 ml of 20 mM Tris buffer (pH 7.5) containing 5 mM MgCl2 and 50 mM NaCl. The amount of label remaining on the filters, which reflects the GTPase activity of Ras, was then determined by liquid scintillation counting in 10 ml of ScintiVerse II (Fisher).

RESULTS Preliminary studies. Initial experiments were designed to gain preliminary evidence for a physical association between GAP molecules and lipids. The first experiment involved cosedimentation of GAP with phospholipid liposomes by centrifugation on a bench top centrifuge. In a pilot experiment, when liposomes composed of 32P-labeled PA were centrifuged in this way (see Materials and Methods), essentially all radioactivity was detected in the pellet. Previous

VOL . 1 l, 1991

experiments confirmed that liposomes rather than micelles result from this preparation procedure. Crude mouse brain extract containing a high level of GAP activity was then incubated with either PA or PC liposomes. After centrifugation, the GAP activities in the supernatants and pellets were assayed. GAP activity remained in the supernatant of brain extract treated with liposomes of PC but was not found in the supernatant with PA. This is consistent with the prediction that GAP molecules associated with PA (which is able to inhibit GAP activity) and was removed with it during centrifugation, whereas PC (which does not inhibit GAP activity) was unable to bind and therefore to remove GAP molecules from solution (data not shown). As a second preliminary line of evidence for an association between GAP and lipids, purified bovine brain GAP (7) was incubated with liposomes as described above. The liposomes were then pelleted by centrifugation as described previously, and GAP molecules were detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. With this approach, GAP molecules were detected in the pellet along with the liposomes of PA and phosphatidylinositol monophosphate (PIP). The purified GAP molecules, however, remained in the supernatant following incubation and centrifugation with PC, PS, or PE liposomes (data not shown). The results indicate that, as described above, GAP binds to PA and PIP liposomes but fails to bind to PC, PS, and PE liposomes. Analysis by size exclusion chromatography. The preliminary analyses outlined above suggested that there is a physical association between those lipids which are able to inhibit GAP activity and the GAP molecule. To more rigorously demonstrate this to be the case, the ability of GAP molecules to associate with mixed lipid micelles was assayed by gel filtration (11). In this approach, the lipid to be tested is dissolved in a mild detergent. The test lipid therefore becomes associated with the detergent micelles. The elution characteristics of the lipids on a gel filtration column are therefore determined by the characteristic size of the detergent micelles. If GAP molecules are able to form a tight association with the lipid contained in a miceile, the GAP migrates as a complex with a detergent micelle. In the studies reported below, the micelles were composed of Triton X-100. There are two facts that complicate this analysis. First, the mixed Triton miceiles have elution characteristics similar to those of the GAP molecules on the Sepharose 4B-200 column that was utilized (as determined in studies with labeled lipids). In addition, the elution characteristics of GAP obtained from living cells are altered by association with other cellular molecules (6, 13, 19). It was therefore necessary to obtain GAP free of intermolecular associations and then to design an experimental system in which the elution of free GAP molecules could unequivocally be distinguished from that of GAP associated with lipid micelles. Free GAP molecules were obtained from a bacterial expression system and partially purified (see Materials and Methods). To rigorously analyze alterations in elution characteristics, purified immunoglobulin molecules were added to each sample as an internal standard. Free GAP molecules would elute slightly later than immunoglobulin, while GAP associated with a typical Triton-mixed micelle would elute earlier than immunoglobulin. Partially purified bacterial GAP molecules were incubated for 1 h with a solution containing 150 p.g of test lipid per ml in 1% Triton X-100 prior to passage over a Sepharose 4B-200 column (1 by 40 cm). Previous studies indicated that this concentration of detergent does not alter the biochemical

ASSOCIATION OF GAP WITH LIPIDS

2787

properties of GAP or its interaction with lipids (30). The abilities of the following two lipids to associate with GAP were compared: arachidonic acid (a 20-carbon fatty acid with four double bonds; 20:4), which is able to inhibit GAP activity, and arachidic acid (20:0), which is inactive against GAP. These two lipids differ only in their degrees of saturation. Passage over the column required approximately 70 min. Individual column fractions were collected and tested by Western analysis with the antipeptide GAP antibody no. 677. In addition, the presence of the immunoglobulin was analyzed by a separate series of Western analyses. The results clearly show that the GAP incubated with and chromatographed in the presence of detergent with arachidonic acid elutes earlier than GAP in the presence of the inactive arachidic acid. The position of the immunoglobulin added as an internal control appeared with a peak in fraction 23 in both samples. The GAP molecules in the presence of arachidic acid eluted with a peak between fractions 23 and 24, slightly later than the immunoglobulin, as would be expected if no association between GAP and the micelles had occurred. In the presence of mixed micelles containing the inhibitory arachidonic acid, however, the apparent size of the GAP molecules increased and appeared in fractions 18 and 19 (Fig. 1). These results indicate that the GAP molecules had associated with arachidonic acid-containing mixed micelles during the chromatographic procedure and that this association was not observed with the inactive but structurally related arachidic acid. The above experiment was repeated numerous times. In some cases, two peaks of GAP molecules were detected in the arachidonic acid-containing sample, one with a mobility increased with respect to that of immunoglobulin and a second with a mobility equal to or just less than that of immunoglobulin. The second peak comigrated with the GAP chromatographed in the presence of the inactive arachidic acid. Incubating longer prior to chromatography and increasing the concentration of the added test lipid from 100 to 150 ,ug/ml appeared to reduce the amount of the more slowly migrating peak. We interpret these observations to indicate that incomplete association of the GAP with the lipid occurs with shorter incubation times and less lipid. Alternately, it is possible that in some preparations a proportion of GAP molecules are inactive and unable to associate with lipid. Retention of GAP by an affinity column. The results above demonstrate that GAP is able to associate with mixed micelles containing arachidonic acid during chromatography through a size exclusion column. To extend these studies, affinity columns containing phospholipid were prepared. Arachidonic acid was activated with carbodiimide and incubated with AFFI-GEL 102 resin, resulting in the chemical linkage of the carboxyl group of arachidonic acid to the resin. This material was then treated with chloroform to remove all unbound arachidonic acid. Over this column was passed 1 ml of GAP-containing cell extract. The eluted fractions were tested for GAP molecules by Western blotting (with polyclonal rabbit antibody no. 663 against the carboxyl terminus of GAP). No reduction in the amount of GAP passing through the column was observed, indicating that the column had not bound GAP molecules efficiently (data not shown). Failure to bind GAP by this column was possibly due to the chemical modification of the carboxyl terminus of the arachidonic acid and thus the destruction of its GAP-binding activity. In support of this possibility, it was previously found that inhibition of GAP activity is strictly dependent upon the structure of the fatty acid (30). Furthermore, with the polar group oriented toward the solid support

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MOL. CELL. BIOL.

A

GAP-" 16 17

18

19

20

21

22

23

24

25

26

27 28

29 30

Fr. No.

B

GAP16 17 18

19

20

21

22

23

24

25

26

27

28

29

Fr. No. FIG. 1. The association of GAP molecules with mixed micelles. Full-length bacterially synthesized GAP (10 ,ug, partially purified as described in Materials and Methods) was incubated with a solution containing 150 ,ug of arachidonic acid (A) or arachidic acid (B) per ml. The lipid was prepared as a mixed micelle in the presence of 1% Triton X-100 (see Materials and Methods), with rat immunoglobulin (Sigma) as an internal standard. The lipid-GAP solutions were then separately passed over a Sepharose 4B-200 size exclusion column which had been equilibrated in running buffer (20 mM Tris-HCl, 5 mM MgCl2, 2 mM dithiothreitol, 1% Triton X-100, 0.15 M NaCl, and 1 mM phenylmethylsulfonyl fluoride [as a protease inhibitor]). Fractions of 300 p.1 were collected and analyzed by PAGE followed by Western analysis with a carboxyl-terminal anti-GAP, antipeptide antibody to determine the mobility of GAP molecules. Separate Western analysis employed antibodies (Boehringer) against the rat immunoglobulin (Sigma) used as an internal standard. The immunoglobulin standard appeared as a peak in fraction 23 in analyses with either arachidonic or arachidic acid. The GAP was observed in fractions 23 and 24 in the presence of arachidic acid (B), as expected for free GAP molecules, which would have a mobility slightly slower than that of immunoglobulin. In the presence of the inhibitory arachidonic acid (A), however, the mobility of the GAP molecules was increased, with a peak in fractions 18 and 19. Under these circumstances, the GAP molecules apparently associated with the arachidonic acid-containing lipid micelles, resulting in increased mobility. The lipid micelles had a mobility similar to that of free GAP, as detected by adding labeled lipids.

and the hydrophobic end of the molecule oriented toward the aqueous phase, the conjugated fatty acid would be inverted with respect to its orientation in natural membranes. To overcome the possible problems associated with modification and orientation of the bound lipid, we designed a related affinity column. In this case, the chemically linked lipid was used to hydrophobically bind and therefore immobilize a second lipid. This second lipid would be unmodified, and because of its interaction with the chemically bound molecules it would be oriented with its polar head group exposed to the aqueous phase as expected for membrane lipids. The proper orientation of the subsequently added lipid would be obtained whether the lipid associated with the chemically bound lipid to form a single bilayer or the bound lipid associated with and retained intact liposomes of the second lipid. To construct this type of affinity column, arachidic acid was chemically conjugated to AFFI-GEL 102 resin. After the removal of unbound lipid with chloroform extraction, liposomes were passed through the AFFI-GEL 102 column containing immobilized arachidic acid. Liposomes of PS, PE, PC, PA, and PIP were found to be retained on the column as expected, presumably because of hydrophobic interaction between the added liposomes and the bound arachidic acid. When mouse brain homogenate was applied to such a column containing PC as the second lipid, GAP was detected in the eluant, suggesting that it had not been retained on the column (Fig. 2A, lane 1). With a PAcontaining column, however, no GAP was detected in the eluant when GAP-containing cell extract was passed over the column (Fig. 2A, lane 2). To confirm that GAP had been retained on the PA affinity column, this column was then

extracted with SDS. The release of GAP from the affinity resin following SDS extraction supports the suggestion that GAP physically associated with the immobilized PA (Fig. 2A, lane 3). SDS-PAGE was also used to analyze the proteins which were not retained by the PA- and PC-containing affinity columns. In both cases, it was apparent that most cytosolic proteins were detected in eluant regardless of phospholipid bound to the column (Fig. 2B, lanes 1 and 2). Both columns were then treated with SDS to release all bound proteins, which were then analyzed on a polyacrylamide gel stained with Coomassie blue. Only a limited amount of protein was retained by and eluted from either the PA or PC column (Fig. 2B, lanes 3 and 4). Because the amount of protein retained by the PA-containing affinity column was limited, the quantitative retention of GAP was apparently highly specific. While few proteins other than GAP were efficiently retained on the PA column, a 55-kDa protein was observed to bind specifically to PA-containing columns (Fig. 2B, lane 4). This protein is apparently unrelated to GAP since it does not bind any of the GAP antisera tested. By using the liposome affinity column, we tested the abilities of several lipids and lipid mixtures to retain GAP. As shown in Table 1, affinity columns with PC, PS, PE, and arachidic acid were totally unable to bind GAP, whereas those with PA, PIP, and arachidonic acid bound GAP efficiently. Mixed lipid preparations with ratios similar to those of natural cell membranes (PC:PE:PS = 2:2:1) failed to bind GAP when added to the affinity column unless 30% PA or PIP was included in the lipid mixture (Table 1). These results are consistent with the gel filtration analysis described above and strongly suggest that GAP physically

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TABLE 1. Retention of GAP by AFFI-GEL 102 column containing various lipidsa Phospholipid(s)*

2

3

Retention

+ PA + Arachidonic acid Arachidic acid PC PC (2 mg) + PE (2 mg) + PS (1 mg) + PC (2 mg) + PE (2 mg) + PS (1 mg) + PIP (2 mg) + PC (2 mg) + PE (2 mg) + PS (1 mg) + PA (2 mg) a An AFFI-GEL 102 column containing various phospholipid liposomes was prepared as described in Materials and Methods. GAP in the form of crude cellular lysate was applied, eluted with EDTA, and detected by Western

1

(A)

-18O 4

blotting.

.-

36 '27

4

3

2

1

(B) affinity columns. (A) Five hundred lysate preparation was passed over an affinity column composed of PC (lane 1) or (p-arachidonyl, -y-stearoyl) PA (lane 2). The columns were then washed with 6 ml (3 column volumes) of washing buffer (20 mM Tris, 5 MM MgCl2, 50 mM NaCI). The presence of GAP molecules was then assessed by Western blotting following SDS electrophoresis. GAP was found only in the wash fraction of the PC affinity column (lane 1). To FIG.

2.

Binding of GAP

to

microliters of crude cellular

ensure

that the absence of detectable GAP in the PA column wash

(lane 2)

was

due to its retention

by

the column, this column

was

proteins. The material released by SDS treatment was then tested. Westemn blotting clearly indicated that GAP had been retained by the PA affinity column and released only after SDS treatment (lane 3). Molecular weight markers (not shown) confirm that the bands shown represent full-length boiled with 1% SDS to release all bound

GAP molecules (116 kDa). (B) Since it is clear from the above

experiment that GAP is retained only by the PA affinity column, it important to determine the specificity of GAP binding. For this analysis, 0.5 ml of crude lysate was passed through a fresh affinity column containing PC or PA. Mfter washing as above, the nonretamned proteins from the PC column (lane 1) and the PA affinity column (lane 2) were resolved by SDS-PAGE followed by Coomassie blue staining. The proteins retained by each column were then released by treatment with SDS as described above (PC was

column, lane 3; PA column, lane 4). The proteins obtained in each of

displayed by SDS-PAGE followed by It is apparent that most cytoplasmic proteins pass through both these columns and that the few proteins which are retained in the two columns are similar. The binding of GAP by the PA-containing column, therefore, is highly specific.

these fractions

staining

were

then

with Coomassie blue.

specifically with PA, PIP, and arachidonic acid. As noted above, the phospholipids that associate with GAP are those with the ability to block GAP activity (30). To expancf results with lipid affinity columns, another type of affinity column was constructed by immobilizing arachidonic acid on Bio-Gel P-2 polyacrylamide beads (which have covalently bonded polyethyleneimine on their surfaces [BioRad Affi-Gel 731]) by ionic interaction. H]arachidonic acid was used to confirm lipid binding to this column. This type of column would be expected to bind the carboxyl group by associates

[3

ionic interaction. The fatty acid molecules which directly associate with the gel matrix, however, would then be able to associate with subsequently added micelles or liposomes of arachidonic acid by hydrophobic interactions. These hydrophobically bound molecules would then most likely be able to associate with GAP molecules. As expected, GAP was retained by Affi-Gel 731 containing immobilized arachidonic acid. The bound GAP was released along with the immobilized lipid following treatment with 1 M NaCl (data not shown). Elution of GAP from PA affinity column by EDTA. Previous studies were unable to demonstrate the reversibility of GAP inhibition by lipids. This possibility could be tested, however, if GAP molecules bound to a lipid affinity column could be released under mild conditions. Efforts to elute GAP from a PA-containing affinity column with high salt (0.5 M NaCl), low pH (pH 5.0), or physical sonication were totally ineffective. This fact suggests that the GAP had not been retained on these columns by simple ionic interactions. In the presence of EDTA, on the other hand, GAP was immediately and quantitatively released from a PA-containing column (Fig. 3). To extend this observation, crude cell extract was applied to a PA affinity column in buffers containing either 5 mM EDTA (Fig. 3A) or 5 mM MgCl2 (Fig. 3B). GAP was retained, as expected, on the column containing MgCl2 but was detected by Western analysis in the flow-through fractions of the EDTA-containing column. Even after extensive washing, the GAP remained bound to the MgCl2-containing column until 10 mM EDTA was added, whereupon GAP was immediately released (Fig. 3B). This result suggests that the association of GAP with the lipid affinity column was dependent upon magnesium ions. We have not determined whether calcium ions would also support lipid-GAP association. A 60-kDa protein from the crude cell lysate which was stained by carboxyl-terminal GAP antibody (no. 663) in Western analysis was presumably a proteolytic fragment of GAP. This protein also associated with the affinity column in a magnesium ion-dependent manner. The inhibition of GAP activity by phospholipid is reversible. The successful elution of GAP from the PA-containing lipid affinity column by EDTA provided the means to test for reversibility of GAP inhibition by lipids. The GAP initially retained on a PA-affinity column was eluted with EDTA and tested for its ability to stimulate Ras GTPase activity in a filter binding assay. For this assay, [-y-32P]GTP is bound to Ras protein, and GTPase activity results in the release of bound label. The presence of GAP activity is therefore indicated by a reduction of bound label. In this experiment,

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A

5

10

15

20

25

Elution Volume (ml)

28

EDTA

4

B

15

10

20

25

28

Elution Volume (ml) FIG. 3. Elution of GAP and GAP activity from a PA affinity column by EDTA. (A) GAP was applied to a PA affinity column in the presence of 5 mM EDTA (with 20 mM Tris, 2 mM dithiothreitol, and 50 mM NaCl [pH 7.5]). Subsequent column fractions were analyzed for GAP by SDS-PAGE and Western analysis. Under these conditions, GAP was not retained by the affinity column and appeared in the first few fractions. (B) GAP was applied to a PA-containing affinity column in the presence of 5 mM MgCl2 or the solution described above. After extensive washing with this buffer, 10 mM EDTA was added at the fraction indicated and elution continued. PAGE and Western analysis indicated that GAP was retained by the column until it was released immediately following EDTA treatment. Molecular weight markers (not shown) confirm that the bands shown represent full-length GAP molecules (116 kDa).

GAP activity was observed immediately after the addition of EDTA to the column, coincident with the appearance of GAP molecules as detected by Western analysis (Fig. 4). This result indicates that the binding of GAP to PA liposomes does not permanently block its biochemical activity. The successful elution of active GAP from the PA affinity column might provide a useful step in GAP purification. Our preliminary results showed that substantial although incomplete purification of GAP was obtained by a single PA liposome affinity column (data not shown). DISCUSSION

Cellular Ras proteins play a critical role in cellular proliferation. Studies with injected anti-Ras antibodies suggest that the proliferative signal initiated by an activated growth factor receptor is received by Ras, which then transfers the signal into the cell to initiate cellular proliferation (25). Recent microinjection studies with two dominant inhibitory Ras mutants have confirmed the earlier results with injected antibody. Microinjection of either inhibitory Ras mutant or the anti-Ras antibody blocked mitogenesis in the presence of serum growth factors or tyrosine kinase containing oncogenes (24a). In an attempt to biochemically characterize the proliferative signal transmitted from growth factor receptors to cellular Ras, we observed that microinjection of anti-Ras antibody was able to block the mitogenesis induced by certain mitogenic lipids with unexpected efficiency (32). The data were consistent with the possibility that lipid metabolism immediately precedes the activation of Ras and that the activity of Ras might therefore be regulated by a lipid. To test the hypothesis that lipids control Ras activity, studies were performed to determine whether lipids might alter the rates of GTP hydrolysis or nucleotide exchange for the Ras protein. While lipids did not directly affect the biochemical properties of Ras, certain lipids inhibited GAP activity in vitro (30). Phospholipids may alter the nucleotide binding status of Ras (and hence its biological activity) by inhibiting GAP. Among the phospholipids that have the most

potent ability to block GAP activity are the phospholipids whose production is altered by mitogenic stimulation (e.g., PA, PIP, and arachidonic acid). In order to obtain evidence that the inhibition of GAP activity by lipids has biological importance, several analyses have been performed. First, the interactions of two Rasrelated proteins with their respective GAP molecules were shown to be inhibited by lipids, suggesting that lipid inhibition of GTPase-activating proteins might be a general phenomenon (28). Second, lipids able to inhibit GAP were identified in mitogen-stimulated cells. They were produced within the first few minutes of mitogenesis and were not observed in contact-inhibited cultures (33). In order to gain further evidence that the activity of GAP molecules might normally be influenced by cellular lipids, studies were undertaken to determine whether lipids physically associate with GAP molecules. If lipids normally function to regulate the activity of GAP, it might be predicted that there would be an identifiable physical interaction between them. Preliminary studies involving low-speed sedimentation of liposomes together with GAP suggested that both GAP activity and GAP molecules cosedimented only with those lipids able to inhibit GAP activity. The preliminary results prompted two types of further analysis. In the first of these, GAP molecules and mixed detergent micelles containing test lipids were cochromatographed on a molecular exclusion column. This experiment was designed to avoid several inherent difficulties in applying this type of analysis to the study of GAP. First, Triton X-100 micelles and GAP molecules have similar elution properties. The association of GAP with a micelle would, therefore, be expected to result in a definite but subtle alteration in its chromatographic characteristics. To allow a conclusive demonstration of altered mobility, immunoglobulin was employed as an internal standard during chromatography. Immunoglobulin (160 kDa) would elute slightly earlier than free GAP (116 kDa). If GAP were to associate with a lipid micelle, however, it would be expected to elute earlier than the immunoglobulin molecules (which would not themselves

ASSOCIATION OF GAP WITH LIPIDS

VOL. 1 l, 1991

c0J

CD

10

'D

m

5

0.

Ct

C) 22

25

27

Fraction No. FIG. 4. Reversibility of GAP inhibition by lipid. A crude cytoplasmic lysate solution containing GAP was passed over a PA affinity column which was washed extensively and eluted with EDTA as described in the legend to Fig. 3. Fractions eluted from the column beginning just prior to EDTA treatment were analyzed by PAGE and Western analysis for GAP molecules. The photograph (top) is of the resulting Western blot. Each fraction was then assayed for GAP activity in the filter binding assay (where reduced remaining label associated with Ras protein indicates a greater rate of GTPase activity). The line drawing (bottom) is positioned such that the point indicating counts remaining after GTPase assay for each fraction is positioned directly beneath the region of the Western analysis corresponding to that fraction. It is clear that both GAP molecules and GAP activity coeluted in this analysis. This result indicates that GAP molecules that were initially immobilized on the PA affinity column can be released from their association with lipid in a biochemically active form. PA binding does not, therefore, irreversibly inactivate GAP activity, even though GAP is not active in the presence of this lipid. Molecular weight markers (not shown) confirm that the bands shown represent full-length GAP molecules (116 kDa). Sufficient Mg2" was added to be in excess of the amounts of EDTA in all fractions. Statistical data were calculated on duplicates of this single determination, but the overall result has been observed repeatedly.

be expected to associate with the lipids). The mobility of

GAP was therefore compared with that of immunoglobulin. The second problem involved in the analysis of GAP molecules is the fact that GAP is commonly associated with other molecules of 60 or 190 kDa in the cell (6). The resulting complexes would migrate in molecular exclusion chromatography more rapidly than the immunoglobulin standard even in the absence of association with lipids. To avoid this problem, free GAP molecules were partially purified from a bacterial expression system. It has been shown previously (30) that these bacterial molecules are inhibited by lipids, as is cellular GAP, but they would not be associated with other eukaryotic proteins. To avoid the possibility that the mobility of GAP molecules during molecular exclusion chromatography is nonspecifically altered by the mixed micelles, control experiments were performed with a structurally related but biochemically inactive lipid. Thus, the mobilities of GAP were compared in mixed micelles of arachidonic acid (which is able to inhibit GAP activity) and arachidic acid, a fatty acid with the same number of carbons as arachidonic

2791

acid but without double bonds or the ability to inhibit GAP activity. Under these conditions, it was clear that GAP associated specifically with mixed micelles composed of arachidonic acid. The second line of evidence that lipids specifically associate with GAP molecules involved the generation of lipid affinity columns. These were constructed first by chemically attaching the carboxyl group of arachidic acid to a solid matrix. To this column were added liposomes of a second lipid. The second lipid was retained by the column, presumably through hydrophobic interactions with the conjugated arachidic acid. It was expected that the second lipid would be positioned in such columns with the polar head groups oriented toward the aqueous phase, as in natural membranes, and would therefore be able to interact in a normal way with GAP molecules. Such columns, prepared with a variety of lipids known to inhibit GAP activity, were able to quantitatively retain the GAP molecules from a crude cytoplasmic lysate (as determined by Western analysis with anti-GAP antibodies). Lipids previously shown to be unable to inhibit GAP activity were unable to retain GAP when utilized in these affinity columns. It was further shown that few proteins in the cytoplasmic lysate were retained by any of the lipid affinity columns tested. Proteins retained by columns composed of PA or PC were essentially indistinguishable as revealed by PAGE, except for a single band at 55 kDa retained specifically by PA which did not react with GAP antibody. Yet the GAP protein was quantitatively retained by the PA-containing column, as detected by Western analysis, but had no observable association with the PC column. The retention of GAP by the PA column, therefore, was highly specific. A number of treatments were employed to remove the GAP bound to a PA affinity column. Only treatment with EDTA released the bound GAP, indicating a requirement for magnesium ions in the association between GAP and lipids. (Further study will determine if calcium ions will substitute for magnesium ions.) The failure of high salt and low pH to release the bound GAP, together with the structural requirements for lipids with binding activity, indicates that specific lipid interactions rather than ionic interaction were responsible for retaining GAP on these columns. When GAP was released from these columns, it had the ability to stimulate the GTPase activity of Ras protein, demonstrating that the association with lipid does not irreversibly inactivate GAP. The fact that the amount of GAP activity released from the lipid affinity columns was often low compared with the amount of activity added to the column is most likely due to the extended manipulation of the protein and resulting nonspecific denaturation. These lipid affinity columns have also been utilized to identify a second protein with the ability to alter the GTPase rate of Ras proteins (29). This protein was released slightly more rapidly than GAP by EDTA treatment. Because its activity on Ras is the opposite of that of GAP and comparatively weak, it was observed only in larger affinity columns where it could be separated from GAP. This protein is able to inhibit the endogenous GTPase rate of Ras protein. In this activity, it is stimulated by many of the same lipids shown previously to inhibit GAP activity (29). This observation further supports the possibility that lipids are involved in the control of cellular Ras activity. Interestingly, the phospholipids that block the activity of and physically associate with GAP are among the phospholipids whose production is stimulated by growth factors. Previous reports have shown that growth factors stimulate

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TSAI ET AL.

the incorporation of labeled phosphate into PA and phosphatidylinositol phosphates (3, 16, 21, 22). Serum has been reported to stimulate the production of arachidonic acid in different cell types (8-12, 14, 15, 23). The mechanism by which the cell produces these lipids in response to mitogenic stimulation is presently unknown. There are clues, however, suggesting that growth factor receptors with tyrosine kinase activity directly interact with enzymes that may alter lipid metabolism in response to mitogenic stimulation. For example, lipocortins and calpactins, potential inhibitors of phospholipase A2, have been shown to be substrates of growth factor receptor (4). In addition, the transforming gene product of Rous sarcoma virus (pp60vsrc) and other membrane-associated tyrosine kinases have been shown to associate with phosphatidylinositol kinase and diacylglycerol kinase (8, 15, 26, 31). More recently, plateletderived growth factor and epidermal growth factor receptors have been shown to associate with phospholipase C and phosphatidylinositol kinase (17, 18). These results may suggest that growth factor receptors are able to alter lipid metabolism by directly interacting with the enzymes involved in lipid metabolism. ACKNOWLEDGMENTS We thank Carson Loomis for evaluation of the data and help in designing experimental protocols and Mark Marshall for providing the GAP expression plasmid. This work was supported in part by grants from the PHS (48662) and from the DeBartolo Fund. REFERENCES 1. Adari, H., D. R. Lowy, B. M. Willumsen, C. J. Der, and F. McCormick. 1988. Guanosine triphosphatase activating protein (GAP) interacts with the p21 effector binding domain. Science 240:518-521. 2. Barbacid, M. 1987. ras genes. Annu. Rev. Biochem. 56:779-827. 3. Bocckino, S., P. F. Blackmore, P. B. Wilson, and J. H. Exton. 1987. Phosphatidate accumulation in hormone-treated hepatocytes via a phospholipase D mechanism. J. Biol. Chem. 262: 15309-15315. 4. Brugge, J. S. 1986. The p35/p36 substrates of protein-tyrosine

kinases as inhibitors of phospholipase A2. Cell 46:149-150. 5. Cale's, C., J. F. Hancock, C. J. Marshall, and A. Hall. 1988. The cytoplasmic protein GAP is implicated as the target for regula-

tion by the ras gene product. Nature (London) 332:548-551. 6. Ellis, C., M. Moran, F. McCormick, and T. Pawson. 1990. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature (London) 343:

377-381. 7. Gibbs, J. B., M. D. Schaber, J. Allard, I. S. Sigal, and E. M.

Scolnick. 1988. Purification of ras GTPase activating protein 8. 9.

10.

11.

from bovine brain. Proc. Natl. Acad. Sci. USA 85:5026-5030. Graziani, Y., E. Erikson, and R. L. Erikson. 1983. Evidence that the Rous sarcoma virus transforming gene product is associated with glycerol kinase activity. J. Biol. Chem. 258:2126-2129. Grillone, L. R., M. A. Clark, R. W. Godfrey, F. Stassen, and S. Crooke. 1988. Vasopression induces V1 receptors to activate phosphatidylinositol- and phosphatidylcholine-specific phospholipase C and stimulates the release of arachidonic acid by at least two pathways in the smooth muscle cell line A-10. J. Biol. Chem. 263:2658-2663. Habenicht, A. J. R., J. A. Glomset, W. C. King, C. Nist, C. D. Mitchell, and R. Ross. 1981. Early changes in phosphatidylinositol and arachidonic acid metabolism in quiescent Swiss 3T3 cells stimulated to divide by platelet derived growth factor. J. Biol. Chem. 256:12329-12335. Hannun, Y. A., C. R. Loomis, and R. M. Bell. 1986. Protein kinase C activation in mixed micelles; mechanistic implications

MOL. CELL. BIOL. of phospholipid, diacylglycerol, and calcium interdependencies. J. Biol. Chem. 261:7184-7190. 12. Hong, S. L., and D. Deykin. 1979. Specificity of phospholipases in methylcholanthrene-transformed mouse fibroblasts activated by bradykinin, thrombin, serum and ionophore A23187. J. Biol. Chem. 254:11463-11466. 13. Kaplan, D. R., D. K. Morrison, G. Wong, F. McCormick, and L. T. Williams. 1990. PDGF 3-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signalling complex. Cell 61:125-133. 14. Lahoua, Z., M. E. Astruc, and C. dePaulet. 1988. Seruminduced arachidonic acid release and prostaglandin biosynthesis are potentiated by oxygenated sterols in NRK 49F cells. Biochim. Biophys. Acta 958:396-404. 15. Macara, I. G., G. V. Marinetti, and P. C. Balduzzi. 1984. Transforming protein of avian sarcoma virus VR2 is associated with phosphatidylinositol kinase activity: possible role in tumorigenesis. Proc. Natl. Acad. Sci. USA 81:2728-2732. 16. MacDonald, M. L., K. F. Mack, and J. A. Glomset. 1987. Regulation of phosphoinositide phosphorylation in Swiss 3T3 cells stimulated by platelet-derived growth factor. J. Biol. Chem. 262:1105-1110. 17. Margolis, B., S. G. Rhee, S. Felder, M. Mervic, R. Lyall, A. Levitski, A. Ullrich, A. Zilberstein, and J. Schlessinger. 1989. EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling. Cell 57:11011107. 18. Meisenhelder, J., P. G. Suh, S. G. Rhee, and T. Hunter. 1989. Phospholipase C-y is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell 57:1109-1122. 19. Molloy, C. J., D. P. Bottaro, T. P. Fleming, M. S. Marshall, J. B. Gibbs, and S. A. Aaronson. 1989. PDGF induction of tyrosine phosphorylation of GTPase activating protein. Nature (London) 342:711-713. 20. Mulcahy, L. S., M. R. Smith, and D. W. Stacey. 1985. Requirement for ras proto-oncogene function during serum-stimulated growth of NIH3T3 cells. Nature (London) 313:241-243. 21. Pike, L. J., and A. T. Eakes. 1987. Epidermal growth factor stimulates the production of phosphatidylinositol monophosphate and the breakdown of polyphosphoinositides in A431 cells. J. Biol. Chem. 262:1644-1651. 22. Sawyer, S., and S. Cohen. 1981. Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A-431 cells. Biochemistry 20:6280-6286. 23. Shier, W. T. 1980. Serum stimulation of phospholipase A2 and prostaglandin release in 3T3 cells is associated with plateletderived growth-promoting activity. Proc. Natl. Acad. Sci. USA 77:137-141. 24. Stacey, D. W., L. A. Feig, and J. B. Gibbs. Submitted for publication. 24a.Stacey, D. W., M. Roudebush, R. Day, S. D. Mosser, J. B. Gibbs, and L. A. Feig. Submitted for publication. 25. Stacey, D. W., M.-H. Tsai, C.-L. Yu, and J. K. Smith. 1988. Critical role of cellular ras proteins in proliferative signal transduction. Cold Spring Harbor Symp. Quant. Biol. 53:871881. 26. Sugimoto, Y., M. Whitman, L. C. Cantley, and R. L. Erikson. 1984. Evidence that the Rous sarcoma virus transforming gene product phosphorylates phosphatidylinositol and diacylglycerol. Proc. Natl. Acad. Sci. USA 81:2117-2121. 27. Trahey, M., and F. McCormick. 1987. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science 238:542-545. 28. Tsai, M.-H., A. Hall, and D. W. Stacey. 1989. Inhibition by phospholipids of the interaction between R-ras, Rho, and their GTPase-activating proteins. Mol. Cell. Biol. 9:5260-5264. 29. Tsai, M. H., C. L. Yu, and D. W. Stacey. 1990. A cytoplasmic protein inhibits the GTPase activity of H-Ras in a phospholipiddependent manner. Science 250:982-985. 30. Tsai, M.-H., C.-L. Yu, F.-W. Wei, and D. W. Stacey. 1989. The effect of GTPase activating protein upon Ras is inhibited by mitogenically responsive lipids. Science 243:522-526.

VOL. 11, 1991 31. Varticovski, L., B. Drucker, D. Morrison, L. Cantley, and T. Roberts. 1989. The colony stimulating factor-1 receptor associates with and activates phosphatidylinositol-3-kinase. Nature (London) 342:699-702. 32. Yu, C.-L., M.-H. Tsai, and D. W. Stacey. 1988. Cellular ras activity and phospholipid metabolism. Cell 52:63-71. 33. Yu, C. L., M. H. Tsai, and D. W. Stacey. 1990. Serum stimula-

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tion of NIH 3T3 cells induces the production of lipids able to inhibit GTPase-activating protein activity. Mol. Cell. Biol. 10: 6683-6689. 34. Zhang, K., J. E. DeClue, W. C. Vass, A. G. Papageorge, F. McCormick, and D. R. Lowy. 1990. Suppression of c-ras transformation by GTPase-activating protein. Nature (London) 346: 754-756.