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(1983) Cell 34, 581-586. 3. Madaule, P. & Axel, R. (1985) Cell 41, 31-40. 4. Furth, M. E.,Davis, L. J.,Fleurdelys, B. & Scolnick, E. M.. (1982) J. Virol. 43, 294-304.
Proc. Natl. Acad. Sci. USA Vol. 83, pp. 6357-6361, September 1986 Biochemistry

p21 ras proteins and guanine nucleotides modulate the phosphorylation of 36- and 17-kilodalton mitochondria-associated proteins (protein phosphorylation)

J. M. BACKER AND I. B. WEINSTEIN Cancer Center and Institute of Cancer Research, Columbia University, New York, NY 10032

Communicated by Richard Axel, May 13, 1986

We have found that, when isolated rat liver ABSTRACT mitochondria are incubated with [y-32P]ATP, there is phosphorylation of 36- and 17-kDa proteins. These proteins together with their protein kinase(s) are released as a complex by incubation of the isolated rat liver mitochondria at 20°C for 30 min with 10 mM glucose 6-phosphate, 0.5 mM inositol phosphate, or 0.01 mM inositol triphosphate. Phosphorylation of the 36- and 17-kDa proteins in this soluble protein fraction is modulated by p21 proteins encoded by ras oncogenes and synthesized in Escherichia coli via recombinant DNA methods. A normal p21 ras protein stimulates phosphorylation of the 36-kDa protein and inhibits phosphorylation of the 17-kDa protein, whereas two transforming p21 ras proteins inhibit phosphorylation of both the 36- and 17-kDa proteins. Although GDP and 5'-guanylyl imidodiphosphate also influence the phosphorylation of these proteins, we present evidence that the effects of p21 ras protein are not simply due to their bound GDP. This novel system may be useful for further studies on the biochemical functions of the p21 ras proteins.

The family of ras oncogenes transforms cells through the action of a 21-kDa protein termed p21 ras (1, 2). Recent findings indicate that ras oncogenes belong to a superfamily of ras-related genes (3). Closely related proteins are found in normal mammalian cells (4), and ras-related genes have been found in Aplysia (3), yeast (5), Drosophila (6), and Dictyostelium (7). These proteins display significant sequence homology with the a subunits of the known regulatory G proteins (8-10). They also display functional similarity to the G proteins (11) because oftheir ability to bind GTP and GDP and their GTPase activity (12-14). The latter activity is reduced in some but not all of the transforming p21 ras proteins (12-15). Recent studies in yeast suggest that the ras gene products might act by modulating the level of cAMP, but precise functions of p21 ras proteins in normal and tumor cells are not known (16, 17). In this paper we report that several purified p21 ras proteins, synthesized in Escherichia coli via recombinant DNA methods, markedly alter the phosphorylation of a 36and a 17-kDa protein. These proteins are phosphorylated in isolated intact rat liver mitochondria and in a soluble protein fraction released from mitochondria after 30 min of incubation at 20°C in the presence of glucose 6-phosphate, inositol phosphate, or inositol triphosphate.

MATERIALS AND METHODS Preparation of Mitochondria and Release of the Enzyme Complex. Female Sprague-Dawley rats (90-150 days old) were anesthetized with diethyl ether and sacrificed by The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

cervical dislocation; the livers were rapidly excised. Mitochondria were isolated at 40C by differential centrifugation in a pH 7.4 medium containing 0.21 M D-mannitol, 0.07 M sucrose, 1 mM Hepes, 0.1 mM EGTA, and 10 mM phenylmethylsulfonyl fluoride essentially as described by Greenawalt (18). The postmitochondrial supernatant fraction obtained after the first centrifugation at 10,000 rpm (Sorvall SS34) was also saved for protein kinase assay. The mitochondrial pellet was resuspended in the isolation medium, and 1 ml of this suspension was incubated at 20'C for 30 min with various additions (see Results). After this incubation the mitochondria were sedimented at 100,000 x g; the supermatants were carefully removed, and aliquots were stored at -200C. Phosphorylation Assay and Gel Electrophoresis. Phosphorylation reactions that contained the intact mitochondria were performed at room temperature in a standard reaction mixture containing 0.21 M D-mannitol, 0.07 M sucrose, 1 mM Hepes (pH 6.8), 2.5 mM MgCl2, 10 mM potassium succinate, rotenone (Sigma) at 2 /,g/ml, oligomycin (Sigma) at 2 ,ug/ml, carboxyatractyloside (Sigma) at 5 ,ug/ml, and mitochondrial protein at 100 ,ug/ml. Phosphorylation reactions that contained only the soluble protein fraction dissociated from mitochondria by the above-described methods were performed in a standard reaction mixture containing 0.21 M D-mannitol, 0.07 M sucrose, 1 mM Hepes (pH 6.8), 2.5 mM MgCl2, 10 mM NaF, and proteins at 10 ,ug/ml, with or without the addition of guanine nucleotides or p21 ras proteins. Reactions were started by the addition of radioactive [y32P]ATP (>3000 Ci/mmol, Amersham; 1 Ci = 37 GBq) to a final concentration of 20-40 nM. After incubation at room temperature for various times, 50-,lA aliquots were taken from the reaction mixture and placed into tubes containing 30 ,u of sample buffer, which contained 10% (vol/vol) glycerol, bromphenol blue at 1 g/liter, 0.05 M EDTA, 0.05 M EGTA, and 7.5% (wt/vol) NaDodSO4. After the addition of 30 ,ul of 2-mercaptoethanol, the samples were heated for 3 min at 100°C and then analyzed by discontinuous NaDodSO4/polyacrylamide gel electrophoresis (19). The stacking gel was polymerized from 3% acrylamide/0. 15% methylenebisacrylamide in 0.1% NaDodSO4/0.125 M Tris HCl, pH 6.8. The resolving gel was either 10% acrylamide (Fig. 1, lanes 1 and 2) or a linear gradient (7.5-15%) of acrylamide (Fig. 1, lanes 3-6) with an acrylamide/methylenebisacrylamide ratio of 20:1 in 0.1% NaDodSO4/0.375 M Tris, pH 8.3. The electrophoresis buffer was 0.025 M Tris'HCl, pH 8.3/0.192 M glycine/0. 1% NaDodSO4. Prestained molecular weight markers were run on the same gel. After electrophoresis, gels were rinsed with 20o (vol/vol) isopropyl alcohol and 10 mM pyrophosphate and autoradiographed with Kodak X-Omat film for various periods of time at 4°C or at -70°C, depending on the intensity of the bands. The relative densities of the Abbreviation: p[NH]ppG, 5'-guanylyl imidodiphosphate.

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FIG. 1. Phosphorylation of the 36- and 17-kDa proteins under various conditions. Apparent molecular masses in kDa for lanes 1 and 2 (10%6 gels) are indicated on the left; corresponding molecular weights for lanes 3-6 (linear 7.5-10%o gradient gels) are indicated on the right. Lanes: 1, phosphorylated products obtained when intact mitochondria were incubated for 3 min; 2, products obtained with the postmitochondrial supernatant fraction; 3, products obtained with the supernatant fraction from mitochondria preincubated for 30 min at 200C; 4, phosphorylated products obtained with the soluble protein fraction released from mitochondria incubated for 30 min at 200C in the presence of 10 mM glucose 6-phosphate; 5, same as lane 4 but with addition of 1 gM [y-P]GTP (>10 mnCi/mmol, Amersham) instead of [y32P]ATP; 6, same as lane 4 but with preautoradiography treatment of the gel overnight with 15% (vol/vol) glacial acetic acid/15% (vol/vol) methanol to remove acid-labile adducts. For a detailed description of the in vitro phosphorylation system and the NaDodSO4 electrophoresis of the 32P-labeled proteins see Materials and Methods.

bands on the autoradiographs were measured with a Chromoscan 3 Joyce-Loebl densitometer. p21 ras Proteins. Purified ras proteins EC p21 ras ([Gly12, Ala59, Gln6l]ras), EJ p2l ras ([Val12, Ala59, Gln6l]ras), and EL p21 ras ([Val12, Ala59, Leu61]ras) were provided by M. Poe, J. Gibbs, and R. Stein of Merck, Sharpe & Dohme Research Laboratories. These proteins were expressed in E. coli as fusion proteins with an amino-terminal fusion sequence that extended from position 0 to -19 and purified to homogeneity as described elsewhere (12, 20, 21). To remove the GDP usually associated with p21 ras proteins (22), the EC and EJ p21 ras proteins were diluted 3-fold with 7.5 M guanidine chloride/i mM EGTA/5 mM Tris HCl, pH 6.8, and these solutions were dialyzed against a x 100 volume of the same buffer for 6 hr at room temperature and then for 16 hr at 4°C against a x5000 volume of 50 mM Tris HCl, pH 6.8/5 mM MgCl2/1 mM dithiothreitol/0.01% octyl glucoside.

RESULTS Phosphorylation of the 36- and 17-kDa Proteins. When freshly isolated rat liver mitochondria were incubated aerobically for 3 min in the presence of succinate, rotenone (to block NADH-linked electron transport), oligomycin (to inhibit the formation of nonradioactive ATP), carboxyatractyloside (to block uptake of the radioactive ATP into mitochondria), and [y-32P]ATP, phosphorylated proteins were detected that migrated in the NaDodSO4/polyacrylamide gels to positions that correspond to 36 and 17 kDa (Fig. 1, lane 1). With longer exposure to the x-ray film, we also detected faint bands at 107 and 70 kDa. In the absence of

(1986)

carboxyatractyloside, we observed a prominent 47-kDa band (data not shown here). We did not observe phosphorylation of these proteins in the postmitochondrial supernatant fraction (cytosolic proteins) obtained during routine isolation of the mitochondria at 40C (Fig. 1, lane 2). However, we did observe phosphorylation of the 17-kDa protein and weak phosphorylation of the 36-kDa protein when the isolated mitochondria were preincubated for 30 min at 20'C, and the supernatant fraction from centrifugation at 100,000 x g was collected and assayed for phosphorylation in a buffered medium containing [y-32P]ATP (Fig. 1, lane 3). Phosphorylation of the 36-kDa protein was markedly enhanced when this supernatant fraction was obtained by incubating the isolated mitochondria with 10 mM glucose 6-phosphate for 30 min at 200C (Fig. 1, lane 4). This concentration of glucose 6-phosphate corresponds to its intracellular concentration and is known to release mitochondria-bound hexokinase and other proteins (23). Incubation of isolated mitochondria with 1 mM rather than 10 mM glucose 6-phosphate did not~yield a soluble protein fraction that was active in phosphorylation of the 36-kDa protein. Active soluble protein fractions also were obtained when the mitochondria were incubated with either 0.5 mM inositol phosphate or 0.01 mM inositol triphosphate (data not shown here). The latter two compounds are involved in intracellular signal transduction (24), and the concentrations we used were within the physiological range for liver cells (25). When the mitochondria obtained after the above preincubations were pelleted, resuspended, and assayed for phosphorylation, we found that the 17- and 36-kDa phosphoproteins could still be detected, although the intensities ofthe bands were less than those obtained when the mitochondria were not preincubated (data not shown here). Thus, incubation of mitochondria at 20°C alone or with low concentrations of glucose 6-phosphate, inositol phosphate, or inositol triphosphate induces partial release of 17- and 36-kDa proteins and presumably their associated kinase(s) into a soluble protein fraction. In all of the studies described below, we performed our in vitro phosphorylation studies with the soluble protein fraction released from the isolated mitochondria by incubation with 10 mM glucose 6-phosphate. When 1 ,uM [y32P]GTP rather than [y32P]ATP was present in the reaction mixture, we observed phosphorylation of the 36-kDa protein and weak phosphorylation of the 17-kDa protein (Fig. 1, lane 5). We found that, with both ATP and GTP as a substrate, the phosphorylated products of the 36-kDa protein, but not those of the 17-kDa protein, were readily hydrolyzed by mild acid (Fig. 1, lane 6). On the other hand, alkali treatment removed the radioactivity from the 17-kDa protein but not from the 36-kDa protein (not shown here). These results suggested that the phosphorylated products in the 36-kDa proteins were phosphoramidate derivatives of basic amino acids (lysine, arginine, or histidine) (26), while the 17-kDa protein was phosphorylated on the more conventional serine, or threonine residues. Further studies are required to identify the precise products. Effects of Guanine Nudeotides on Phosphorylation of the 36and 17-kDa Proteins. Kinetic studies (see control curves in Fig. 2) indicated that at 20°C phosphorylation of the 36-kDa protein plateaued at -5 min, whereas phosphorylation of the 17-kDa protein proceeded much more rapidly and plateaued within 1 min. Additional studies indicated that after -5 min the level of phosphorylation of both proteins declined, perhaps because of turnover or an associated protein phosphatase activity. Since guanine nucleotides modulate a number of important biologic activities (11), we assessed the effects of GDP in this system. We found that the addition of 80 nM GDP enhanced the initial rate of phosphorylation of the 36-kDa protein but inhibited by about a factor of 2 the

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Proc. Natl. Acad. Sci. USA 83 (1986)

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Time, min FIG. 2. Modulation of the phosphorylation of the 36- and 17-kDa proteins by guanine nucleotides. (A) Phosphorylation of the 36-kDa protein. Control; A, 80 nM GDP; A, 800 nM GDP. (B) Phosphorylation of the 17-kDa protein. 9, Control; A, 80 nM GDP; A, 800 nM GDP. (C) Phosphorylation of the 36-kDa protein; *, control; A, 80 nM p[NH]ppG; A, 800 nM p[NH]ppG. (D) Phosphorylation of the 17-kDa protein. *, Control; A, 80 nM p[NH]ppG; A, 800 nM p[NH]ppG. Phosphorylation reactions were performed as described by using the soluble protein fraction obtained from mitochondria preincubated with glucose 6-phosphate. The relative extents of phosphorylation of the 36- and 17-kDa proteins were determined by densitometry of the autoradiographs. *,

phosphorylation of the 17-kDa protein (Fig. 2 A and B). A 10-fold higher concentration of GDP (800 nM) inhibited the phosphorylation of both proteins (Fig. 2 A and B). A nonhydrolyzable analog of GTP, 5'-guanylyl imidodiphosphate (p[NH]ppG), exerted effects on the phosphorylation of both proteins similar to those seen with GDP (Fig. 2 C and D), providing evidence that guanine nucleotides can modulate these reactions directly without further metabolism. Effects of p21 ras Proteins. In view of the fact that the p21 ras proteins bind guanine nucleotides (12-15) and in view of the current interest in the biochemical functions of these proteins, we assessed the effects of purified p21 proteins on phosphorylation of the 36- and 17-kDa proteins in our in vitro system. We assayed phosphorylation of the 36- and 17-kDa proteins in the presence of several different p21 ras proteins, each of which had been expressed in E. coli as a fusion protein with an amino-terminal fusion sequence extending from -19 to 0 and had been purified to homogeneity (12, 20, 21). These p21 ras proteins contained GDP (22), which binds to these proteins with a dissociation constant of about 108 M'1 (27). At the concentrations of p21 ras proteins used in our experiments (400 nM), we estimated that 10-20% of the ras

bound GDP would be dissociated from the protein at equilibrium. Therefore, the concentration of free GDP would be -80 nM. Our studies described above (Fig. 2) indicated that this concentration offree GDP stimulated phosphorylation of the 36-kDa protein and partially inhibited phosphorylation of the 17-kDa protein. We found that the EC p21 ras protein ([Gly12, Ala59, Gln61]ras), corresponding to the normal product of the human HA-ras oncogene (HRAS in human gene nomenclature) (20), when added at 400 nM to our system, enhanced the initial rate of phosphorylation of the 36-kDa protein -3-fold (Fig. 3A). When tested at 1.2 A.M, this protein also increased the plateau level =2-fold higher than the control value (data not shown here). In contrast, two transforming p21 ras proteins, EJ p2l ras ([Val'2, Ala59, Gln61]ras), corresponding to the transforming p21 protein from a human bladder tumor (28), and EL p21 ras ([Gln12, Ala59, Leu6l]ras), corresponding to the transforming p21 ras protein of a human lung carcinoma (20, 29), strongly inhibited phosphorylation of the 36-kDa protein (Fig. 3A). Both the normal EC p21 ras protein and the two transforming proteins EJ p21 ras and EL p21 ras markedly inhibited by about a factor of 10 the phosphorylation of the 17-kDa protein (Fig. 3B). ras

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The enhanced initial rate of phosphorylation of the 36-kDa protein induced by the EC p21 ras protein might simply be due to release of the GDP associated with this protein, as mentioned above. Therefore, we removed GDP from the EC p21 ras protein by extensive dialysis in 7.5 M guanidine chloride and then assayed the dialyzed protein in our system in the absence or presence of 400 nM GDP or p[NH]ppG. When added alone, 400 nM GDP or 400 nM p[NH]ppG stimulated phosphorylation of the 36-kDa protein and inhibited phosphorylation of the 17-kDa protein (Table 1), consistent with results shown in Fig. 2. We found that the dialyzed EC p21 ras protein retained its ability to stimulate phosphorylation of the 36-kDa protein, and the addition of 400 nM GDP or p[NH]ppG did not further enhance this stimulation (Table 1). On the other hand, dialysis reduced the ability of the EC p21 ras protein to inhibit phosphorylation of the 17-kDa protein, and maximum inhibition was obtained only when 400 nM GDP or p[NH]ppG was added to the reaction mixture containing the dialyzed EC p21 ras protein

(Table 1). Thus, the ability of the EC p21 ras protein to stimulate phosphorylation of the 36-kDa protein is not dependent on bound guanine nucleotides, whereas the inhibitory effect of the EC p21 ras protein on phosphorylation of the 17-kDa protein is maximal in the presence of a guanine nucleotide, which can be either GDP or p[NH]ppG. When we tested the dialyzed transforming EJ p21 ras protein, we found that it caused only partial inhibition of phosphorylation of the 36-kDa protein and no significant inhibition of phosphorylation of the 17-kDa protein (Table 1), in contrast to the marked inhibitions seen with the undialyzed EJ p21 ras protein (Fig. 3). The addition of 400 nM of either GDP or p[NH]ppG to reaction systems containing the dialyzed EJ p21 ras protein resulted in marked inhibition of phosphorylation of both the 36- and 17-kDa proteins, although the two nucleotides differed quantitatively with respect to the extents of inhibition obtained (Table 1).

Table 1. Effects of dialyzed p21 ras proteins alone and in combination with guanine nucleotides on phosphorylation of the 36- and 17-kDa proteins Extent of phosphorylation,

The present studies describe an in vitro system derived from isolated rat liver mitochondria that results in phosphorylation of 36- and 17-kDa proteins. The proteins used in this system appear to be associated with the outer mitochondrial membrane because they are readily released from mitochondria by incubation at room temperature in the presence of physiological concentrations of glucose 6-phosphate, inositol phosphate, or inositol triphosphate. In addition, when we incubated intact mitochondria with [-32P]ATP, we also observed phosphorylation of the 36- and 17-kDa proteins, even in the presence of inhibitors that block the uptake of the labeled ATP (Fig. 1). The 36- and 17-kDa phosphorylated proteins seen in the soluble protein fraction (Fig. 1, lane 4) appear to be the same as those seen with intact mitochondria because of their similar size and acid lability or stability. It is curious that the phosphorylated residues produced on the 36-kDa protein are acid labile. Acid-labile phosphoramide derivatives of basic amino acids have been identified previously in other cellular proteins (26). Additional studies are required to determine whether the acid-labile products on the 36-kDa protein represent transient intermediates in a subsequent phosphorylation reaction or whether they modulate the function of the 36-kDa protein itself. Since the phosphorylated residues formed on the 17-kDa protein are acid stable but alkali labile, they presumably represent the more conventional phosphoserine or phosphothreonine residues.

% of control 36-kDa 17-kDa Additions protein protein None 100 100 GDP 180 10 250 p[NH]ppG 80 470 64 Dialyzed EC p21 ras Dialyzed EC p21 ras/GDP 350 20 Dialyzed EC p21 ras/p[NH]ppG 250 21 96 Dialyzed EJ p21 ras 72 Dialyzed EJ p21 ras/GDP 46 15 Dialyzed EJ p21 ras/p[NH]ppG 10 51 The EC and EJ p21 ras proteins were dialyzed and then added at 400 nM to the phosphorylation system and assayed as described in Fig. 3 and in Materials and Methods. The relative extents of phosphorylation of the 36- and 17-kDa proteins after a 1-min reaction were determined by NaDodSO4 gel electrophoresis and densitometry of the autoradiographs. When GDP or p[NH]ppG (each at 400 nM) was added to the reaction mixture together with the dialyzed p21 ras proteins, the samples were preincubated at 20°C for 5 min before the addition of the other components to the reaction mixture.

DISCUSSION

Biochemistry: Backer and Weinstein A striking finding in the present studies is the ability of purified p21 ras proteins to modulate the in vitro phosphorylation ofthe 36- and 17-kDa proteins (Fig. 3). It is ofinterest that the effects of the normal p21 ras protein, EC p21 ras, are qualitatively different from those of the transforming p21 ras proteins EJ p21 ras and EL p21 ras. Thus, whereas both the normal and the two transforming proteins inhibit phosphorylation of the 17-kDa protein, the normal protein stimulates, but the transforming proteins inhibit, phosphorylation of the 36-kDa protein (Fig. 3). It is unlikely that these effects are simply due to the GDP that is bound to these proteins and its dissociation in our reaction system, since this would not explain the qualitative difference between the effects of the normal and transforming p21 ras proteins. Furthermore, when the EC p21 ras protein was dialyzed to remove bound GDP, it still enhanced the phosphorylation of the 36-kDa protein, and this enhancement was not augmented by supplementation of the reaction system with GDP or p[NH]ppG. At the same time it is also apparent that maximal activity of the EJ p21 ras and EL p21 ras proteins required the presence of GDP (Table 1) and that free GDP and p[NH]ppG are capable of stimulating phosphorylation of the 36-kDa protein and inhibiting phosphorylation of the 17-kDa protein (Fig. 2 and Table 1). The fact that p[NH]ppG is also as active as GDP provides evidence that the action of the guanine nucleotide does not require further metabolism. Perhaps, the soluble protein fraction used in our reaction system also contains a guanine nucleotide-binding protein that regulates phosphorylation of the 36- and 17-kDa proteins, and the p21 ras proteins that we add to this system may play an analogous role. Further studies are required to clarify the biological significance of our findings. Nevertheless, this subcellular system could provide insights into mechanisms by which normal and transforming p21 ras proteins may influence protein phosphorylation. We are indebted to Dr. M. Poe, J. B. Gibbs, and R. B. Stein of Merck Sharp and Dohme Research Laboratories for providing the p21 ras proteins used in this study and for their valuable suggestions. 1. Ellis, R. W., Lowy, D. R. & Scolnick, E. M. (1982) Adv. Viral. Oncol. 1, 107-126. 2. Taparowsky, E., Shimuzu, K., Goldfarb, M. & Wigler, M. (1983) Cell 34, 581-586. 3. Madaule, P. & Axel, R. (1985) Cell 41, 31-40. 4. Furth, M. E., Davis, L. J., Fleurdelys, B. & Scolnick, E. M.

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