Identification of an intracellular protein that specifically interacts with ...

Proc. Natl. Acad. Sci. USA Vol. 86, pp. 8678-8682, November 1989 Biochemistry

Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein GRACE LEE*t, ZEEV A. RONAItt§, MATTHEW R. PINCUS*§¶, PAUL W. BRANDT-RAUFtIl, RANDALL B. MURPHY* **, THOMAS M. DELOHERYt, SUSUMU NISHIMURAtt, ZIRO YAMAIZUMItt, AND 1. BERNARD WEINSTEINt§'1 *Department of Chemistry, New York University, 4 Washington Place, New York, NY 10003; tComprehensive Cancer Center, Columbia University College of Physicians and Surgeons, 701 West 168th Street, New York, NY 10032; tMolecular Carcinogenesis Program, American Health Foundation, 1 Dana Road. Valhalla, NY 10595; $Department of Pathology, State University of New York Health Science Center at Syracuse, 750 East Adams Street, Syracuse, NY 13210; IlDivision of Environmental Sciences, Columbia University School of Public Health, and Department of Medicine, Columbia University College of Physicians and Surgeons, 60 Haven Avenue, New York, NY 10032; ttBiology Division, National Cancer Center Research Institute, Chuo-Ku, Tokyo, Japan; and **Department of Psychiatry, New York Hospital-Cornell University Medical College, Bourne Laboratory, 21 Bloomingdale Road, White Plains, NY 10605

Communicated by H. A. Scheraga, August 15, 1989

is unclear as to whether GAP is a "target" of the p21 protein-i.e., a possible receptor for this protein-or is a regulatory protein-i.e., one that simply regulates the GTPase activity of the normal protein. It is clearly desirable to search for other proteins with which the p21 protein may directly interact. A recent communication demonstrates that overexpressed nononcogenic Ha-ras p21 protein interacts with a 60-kDa protein in Rat-i cells (7); this protein apparently differs from GAP (7). Several other studies have suggested possible intracellular metabolic effects of oncogenic p21 proteins. In particular, it has been shown that the p21 protein may lead to activation of the phosphotidylinositol pathway (8), may be a substrate for protein kinase C (9), and may promote activation of various mitochondrial enzymes (10). In addition, microinjection experiments suggest that the p21 protein may be activated by proteins encoded by other oncogenes, such as src (11). In this communication, we present the results of experiment-s designed to identify possible protein targets of an oncogenic p21 protein.. The approach, summarized in Fig. 1, was to modify the p21 protein with the bifunctional reagent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) (12), which reacts covalently with proteins mainly at the c-amino groups of lysine residues to form amides (Fig. 1). In these reactions, N-hydroxysuccinimide is liberated. The covalently attached reagent contains the nitrophenylazide group that, upon activation by light, produces a highly reactive nitrene capable of forming covalent bonds with residues in neighboring proteins with which the p2l protein may associate. This photoactivatable derivatized protein was introduced quantitatively into NIH 3T3 cells using a modified erythrocyte (RBC) fusion technique (13). The cells were then exposed to UV-irradiation at a wavelength of 330-350 nm, thereby activating the photoreactive portion of the SANPAH-labeled protein. Immunoprecipitation of modified p21 proteins in lysates of the exposed cells with anti-p21 monoclonal antibodies followed by SDS/PAGE of the immunoprecipitated proteins led to identification of specific cellular proteins associated with the p21 protein.

An oncogenic 21-kDa (p21) protein (Harvey ABSTRACT RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocytemediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with the modified p21 protein, the cells were pulsed with [35S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21-protein complexes. By using this technique, we found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein into the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. We suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes.

The RAS oncogene-encoded p21 proteins, which can be activated by the binding of GTP, are known to play an important role in cell transformation (1-3). Mutant forms of these proteins that cause cell transformation contain sequences that are identical to normal cellular p21 protein products except for amino acid substitution(s) at critical positions, usually at Gly-12 ([Gly12]p21), Gln-61 ([Gln61]p21), and Glu-63 ([Glu63]p21) in the polypeptide chain (1-3). Most of these positions occur in regions of the protein that are involved in the binding of GDP and GTP (4). Certain amino acid substitutions in the region of the protein from residues 32 to 40, a region not involved in nucleotide binding, can inactivate RAS-encoded oncogenic proteins that contain other amino acid substitutions known to cause cell transformation (5). This region has now been implicated in the binding of p21 proteins to a GTPase-activating protein (GAP) (5, 6). The latter protein causes large rate enhancements in the GTPase activity of the normal (nononcogenic) p21 protein but does not cause such rate enhancements in the oncogenic proteins (6), although it binds to both proteins. Currently, it

MATERIALS AND METHODS Materials. SANPAH was purchased from Pierce and was used directly. Bovine serum albumin (BSA), rhodaminedextran (70 kDa), goat anti-rat antibodies conjugated to alkaline phosphatase, a-naphthyl phosphate, and fast red Abbreviations: GAP, GTPase-activating protein; SANPAH, Nsuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate; BSA, bovine serum albumin; RBC, erythrocyte. §To whom reprint requests should be addressed.

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. 8678

Proc. Natl. Acad. Sci. USA 86 (1989)

Biochemistry: Lee et al.

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were purchased from Sigma. Goat anti-mouse IgG-lissamine rhodamine conjugate was purchased from Boehringer Mannheim. Polyethylene glycol 8000 was purchased from Baker. Protein G was purchased from Pharmacia. Normal human [Gly'2]p21 and oncogenic human [Val12]p21 were overexpressed in Escherichia coli employing an expression vector (pGH-L9) containing the chemically synthesized HRAS gene and purified as described (14). The protein concentration in stock solutions as determined by Bradford assay (Bio-Rad) was 3.0 mg/ml. Reaction of [Val'2]p21 with SANPAH. Stock p21 protein solution (600 ,l) described above was incubated with 25.6 mM SANPAH in dimethyl sulfoxide (150 ,l) for 12 hr. This reaction mixture was then transferred into a dialysis bag in the presence of an equal volume of 10 mM NaHCO3 and was dialyzed against 100 mM 2-aminoethanol (pH 8.8) for 24 hr followed by further dialysis against a solution of 120 mM NaCl and 20 mM NaHCO3, for an additional 24 hr at 4°C. The absorbance of the dialyzed solution of SANPAH-modified p21 protein revealed a label to protein ratio of 1:1. This modified protein was used directly in the experiments described below. Cell Fusion, Reaction of Light-Activated [Val'2Jp21 Protein, and Identification of Labeled Protein Products. Introduction of the p21 protein into the NIH 3T3 cells was carried out as described (13). The dialyzed protein (100,ug, except in Fig. 2A) was incubated with 0.5 ml of packed human RBCs in the presence of BSA (400 ,tg). Distilled water was then gradually added (4 parts of H20 to 1 part of packed RBCs) to effect cell lysis (13). This suspension was then mixed by rotation at room temperature for 45 min. The RBCs were then sealed by slowly adding 0.1 vol of 1Ox PBS (1x PBS = 137 mM NaCl/2.7 mM CaCl2/8.1 mM Na2HPO4/1.5 mM KH2PO4). The sealed ghosts containing the modified protein were then sedimented by centrifugation and washed. The packed ghosts (0.5 ml) were then fused with a suspension of 2.5 x 107 NIH 3T3 cells [which had been grown to confluence in Dulbecco's modified Eagle's medium (DMEM) plus 10o (vol/vol) calf serum] in DMEM containing 50% (wt/vol) polyethylene glycol 8000 for 1 min at 25°C. This mixture was then slowly diluted with DMEM. The ratio of RBC ghosts to NIH 3T3 cells was 200:1. By using a goat anti-mouse IgG-lissamine rhodamine complex as a marker to monitor the efficiency of the cell fusion, we found that about 25% of the cells had taken up the dye when examined 24 hr after fusion. This was

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SANPAH reacts with nucleophiles, most probably the E-amino groups of lysine residues, in the p21 protein to form a covalent complex with this protein. (C) The covalently modified p21 protein was then introduced into the NIH 3T3 cells by the RBC fusion method. Once inside the cells, it was subjected to flash photolysis with light at 330-350 nm. The resulting aryl nitrene formed from the azide group reacts with any electron-rich functional group (R) of a protein associated with p21 (protein X) to form a covalent complex in which the p21 protein is covalently linked to protein X by the SANPAH linker.

determined by fluoresence-activated cell sorter (FACS) analysis using a Becton-Dickenson FACS IV instrument. The NIH 3T3 cells were then sedimented and replated in the medium in which they were grown to prevent serum stimulation. The monolayer cultures were then pulse-labeled from 0 to 8 hr after plating by exposure to [35S]methionine (100 ,uCi/ml; 800 Ci/mmol; 1 Ci = 37 GBq; New England Nuclear) for 30 min. Continuous labeling experiments were also done in which cells were exposed to [35S]methionine continuously from the time of plating until irradiation. In either case, at the end of the indicated time (see Fig. 4), the cells were UV-irradiated at 330-350 nm for 30 min at room temperature using a Sylvania UV-B hand lamp. The cells were then scraped from the plates in Furth's lysis buffer (15). This solution was centrifuged at 15,000 rpm for 5 min at 4°C in a TI 50 rotor, and an amount of the supernatant fraction sufficient to contain 1-5 x 101 cpm was immunoprecipitated with the Y13-259 antibody and protein G. The precipitate was collected, the proteins were eluted with SDS loading buffer [125 mM Tris HCl, pH 6.8/4% (wt/vol) SDS/10% glycerol/ 0.02% bromophenol blue/4% (vol/vol) 2-mercaptoethanoli, loaded onto a stacking gel, and separated by SDS/PAGE (8% gel). The gel was then subjected to fluorography (16) to enhance the 35S signal. Autoradiograms of the gels were exposed for at least 1 week prior to analysis. Functional Assays for the Inserted [Val'2Jp21 Protein. To assay the biologic effect of the oncogenic p21 protein that was introduced into the NIH 3T3 cells, we measured changes induced by the protein by assaying for both cellular pinocytosis and DNA synthesis, since both of these properties are known to increase when an oncogenic p21 protein is introduced into NIH 3T3 cells by microinjection (17, 18). Pinocytotic activity of fused cells was assayed by incubating the cells with rhodamine-dextran (1 mg/ml) in DMEM for 10 min at 37°C. The cells were then washed four times in PBS 1% BSA, pH 7.0 and the dye uptake was then measured by flow cytometry. Cellular rhodamine-dextran fluorescence was monitored at various times after fusion and with different concentrations of [Gly12]p21, [Val12]p21, or BSA. All cellular rhodamine fluorescence measurements were made using a FACS IV flow cytometer (Becton-Dickenson) with 514-nm laser light for excitation. Fluorescence was collected using 520-nm and 580-nm long-pass interference filters. Fluorescence measurements were made using linear signal processing electronics with 256-channel resolution. Debris was elim-

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inated from cell-sample measurements using a forwardangle-light-scatter threshold trigger. All fluorescence values represent relative linear mean intensity for each sample. Stimulation of DNA synthesis in these cells was measured by [methyl-3H]thymidine incorporation. In these experiments, the cells were incubated for 1 hr with [methyl3H]thymidine (4 tkCi/ml, 80.9 Ci/mmol; New England Nuclear) at various time points after the RBC fusion step (see Fig. 2B) and were then washed with PBS (pH 7.0), and radioactivity was measured directly. In control experiments equivalent amounts of either nononcogenic [Gly12]p21 protein or BSA replaced the oncogenic [Val12]p21 in the RBC cell fusion procedure.

RESULTS Functional Assays for Oncogenic p21 Protein Using Cell Fusion. We first measured pinocytotic activity by assaying the uptake of rhodamine-dextran particles by NIH 3T3 cells at various time points after the cells were loaded with the p21 protein. These studies revealed a significant stimulation of pinocytotic activity, which was maximal 9 hr after cell fusion (data not shown). Therefore, we used this time point to measure the concentration dependence of pinocytotic activity. Typical results are shown in Fig. 2A. As can be seen in this figure, a marked concentration-dependent enhancement of pinocytotic activity was observed when the cells were fused with RBCs that contained various amounts of the oncogenic [Val12]p21 protein. Only slight stimulation was obtained in parallel studies with the nononcogenic [Gly12]p21 protein when compared to the results obtained with BSA (Fig. 2A). We next assayed the effect of these p21 proteins on DNA synthesis. As shown in Fig. 2B, the [Val12]p21 protein produced a marked stimulation of [3H]thymidine incorporation that reached a peak about 14 hr after introduction of the p21 protein. Stimulation was also seen with the [0ly12]p21 protein, but at all time points, this was only about 50% that obtained with the oncogenic [Val12]p21 protein. The results with BSA (data not shown) were essentially the same as those

obtained with [Gly12]p21 protein. Identification of Cellular Proteins That Bind to the Oncogenic p21 Protein. To identify cellular proteins that might

form a specific complex with the introduced p21 protein and that might, therefore, be involved in mediating its biologic effects, cells that had incorporated the p21 protein tagged with the photoaffinity label were irradiated and lysed at either 25 min or 2.5 hr after the introduction of the protein. Cell extracts were then subjected to immunoprecipitation with an anti-p21 monoclonal antibody (15). The p21 protein and any protein to which it might be covalently bound were then separated by SDS/PAGE and identified by immunoblotting (using the goat anti-rat antibodies conjugated to alkaline phosphatase, a-naphthyl phosphate, and fast red) as shown in Fig. 3. The 25-min and 2.5-hr samples obtained from cells that had received the [Val12]p21 protein (lanes 2 and 1, respectively) revealed three bands of 51, 64 and 82 kDa, which were most prominent in the 2.5-hr sample. The 64-kDa band was much more abundant than the other two bands. Presumably, the 64-kDa complex is composed of the p21 protein bound to a target protein of about 43 kDa. The 64-kDa complex was not seen in the extracts obtained from cells that had received BSA (lanes 5 and 6) or cells that had received unmodified [Val12]p21 protein (lanes 3 and 4). All of the lanes displayed a high molecular mass band at about 150 kDa. This band appears to represent nonspecific interaction with the secondary antibody used in the procedure. Other faint bands were also observed, such as bands at 109 kDa and 46 kDa (e.g., Fig. 3, lanes 1 and 2). These may represent other proteins with which the p21 protein may make contact. To increase the sensitivity of our assays and to obtain detailed kinetic information on the appearance of the protein complex described above, the SANPAH-labeled [Val'12p21 protein was introduced into the cells and at various times cellular proteins were labeled with 135S]methionine. The cells were then irradiated, proteins were extracted and immunoprecipitated with the anti-p21 antibody, and the immunoprecipitate was analyzed by SDS/PAGE and autoradiography to detect specific [35S]methionine-labeled cellular protein-p21 complexes. These experiments also revealed a major 64-kDa protein and trace amounts of other proteins (Fig. 4). In pulse-labeling experiments, the maximal level of the labeled 64-kDa protein complex occurred about 4 hr after the fusion step; this protein complex reached a maximal level at 5-6 hr in the continuous labeling experiments. As can be seen in Fig. 4, lane B6, when BSA was substituted for the p21 protein, no

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FIG. 3. Immunoblot of an NIH 3T3 cell ly(sate obtained after immunoprecipitation with monoclonal anti-RAS3 antibody Y13-259. Cells were fused with RBCs loaded with SANPAH-labeled [Val'2]p21, unlabeled [Val'2]p21, or BSA and irraLdiated, and extracts 5) were prepared 25 min (lanes 2, 4, and 6) or 2.5 hir (lanes 1, 3, IfAArszalnf --II f..^;IwC~th' X-i- ceii Lu pg U t projJin) was L.e IysaLe fusionU. roll aIler tine equivalent oi la immunoprecipitated with Y13-259 (-1:100 dilution) and a staphylococcal protein A-rabbit anti-rat IgG conjugate. The immunoprecipitate was released by boiling in 50 al of sample buffer, and this volume was loaded onto each lane. Lanes: 1 and 2, cells fused with SANPAH-labeled [Val12]p21; 3 and 4, unlabeled [Val12]p21; 5 and 6, BSA.

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labeled proteins were detected in the immunoprecipitate of the cell extract, indicating that formation of the 64-kDa protein complex was specific for the photoaffinity-labeled p21 protein. To verify that the 64-kDa protein complex seen in the above studies was a specific complex with the p21 protein, two further experiments were performed. These were a "preflash" experiment in which the photoaffinity-labeled [Vall2]p21 protein was irradiated at 330-350 nm prior to cell fusion and a "postflash" experiment in which the photoaffinity-labeled [Vall2]p21 protein was irradiated after its uptake into the cells but just prior to incubation of the cells with [35S]methionine (Fig. 5). In both experiments, the photoafP3 P4

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8681

finity label was inactivated prior to its opportunity to associate with 35S-labeled cellular proteins. No radioactive protein complexes were found in the preflash experiment (Fig. 5, lane 3), and only a trace of the 64-kDa complex was found in the postflash experiment (Fig. 5, lane 4). However, the 64-kDa protein complex is clearly seen in Fig. 5, lanes 1 and 2 (4 and 4.5 hr, respectively). The small amount of the 64-kDa band seen in the postflash experiment (Fig. 5, lane 4) may have resulted from ambient light activation of the azide. Quantitative densitometry of the gel shown in Fig. 5 indicated that the 64-kDa band in lane 4 (postflash) contained less than 27% of the radioactivity of the corresponding band seen in lane 2 and no significant density was seen in the corresponding position of lane 3. These results suggest that introduction of the [Val12]p21 protein into the cells actually induces within a few hours the synthesis of a 43-kDa protein that becomes firmly associated with the introduced p21 protein. The low level of the 64-kDa complex at the 25-min point in Fig. 4 is consistent with the de novo appearance of this protein. Other interpretations, however, have not been excluded.

DISCUSSION The purpose of the present experiments was to introduce "reporter" oncogenic p21 protein molecules quantitatively into NIH 3T3 cells to identify putative intracellular proteins with which they might interact. The oncogenic form of the protein was used in these experiments because its biological activity can be easily monitored. These experiments were designed so that covalent labeling reactions could be initiated at desired times by light-induced photolysis. An alternative approach has been developed in which cells that overexpress 1

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-46 - 30 FIG. 4. SDS/PAGE of the immunoprecipitates of NIH 3T3 cell lysates from cells incubated with [35S]methionine showing comparison of pulsed versus continuous labeling. After introduction of SANPAH-labeled [Val12]p21 into the NIH 3T3 cells, [35S]methionine labeling was carried out as a series of 30-min pulses (lanes P) or a continuous labeling (lanes C). Various times are indicated by lane labels (in hr)-i.e., P2 is 2 hr after fusion. Lane B6 is a BSA control at 6 hr. Immunoprecipitates of the cell lysates were prepared with the Y13-259 antibody and analyzed by SDS/PAGE.

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lysates from cells fused with RBCs containing the SANPAH-labeled [Val12]p21 protein. The cells were irradiated 4 (lane 1) and 4.5 (lane 2) hr after fusion. Lane 3 is a control in which the photoaffinity label was inactivated prior to the introduction of the SANPAH-labeled p21 protein into cells. After introduction of the protein into the cells, the cells were pulse-labeled with [35S]methionine at 4 hr for 30 min and

extracts were analyzed. Lane 4 represents a control in which the SANPAH-labeled [Val'2]p21 protein was introduced into cells, the cells were irradiated 4 hr later, and then they were pulse-labeled for 30 min with [35S]methionine, and extracts were processed.

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a nononcogenic p21 protein were treated with a bifunctional cleavable cross-linking agent that links the overexpressed protein to other putative intracellular targets (7). The latter studies identified a 60-kDa protein that is distinct from the proteins GAP and pp6Osrc (7). In the present experiments, three protein complexes, of 51, 64, and 82 kDa, were identified. The 64-kDa complex was the most abundant and reached a maximum level 4-6 hr after introduction of the p21 protein. It is possible that this time course reflects the de novo synthesis of a putative 43-kDa protein or the time needed for the exogenous p21 to be processed intracellularly before it is capable of interaction with the target protein. The introduced p21 protein also produced functional effects in the NIH 3T3 cells, including increased DNA synthesis, as quantified by the incorporation of [3H]thymidine and enhanced pinocytosis. The pinocytotic activity reached a maximum level about 9 hr after introduction of the oncogenic p21 protein. Enhanced DNA synthesis and pinocytotic activity have been described in p21 microinjection studies (17, 18). Since the appearance of the 64-kDa protein complex occurred prior to these functional changes, it may play a role in mediating these changes. The 64-kDa complex is composed of the p21 protein and the protein to which it is covalently linked. The molecular mass of this protein would, therefore, be expected to be 43 kDa. Thus, this protein is different from characterized proteins that interact with p21 (6, 7). It is of interest that a higher molecular mass complex of 82 kDa, also identified in our study, appears to be a complex between the p21 protein and a protein of about 60 kDa. The latter protein may correspond to the 60-kDa protein found to complex with p21 in studies by other investigators (7). Other studies have demonstrated that the p21 protein binds to GAP, a cytosolic pattern (6). It has been suggested that the p21-GAP complex then migrates to the cell membrane since attachment of the p21 protein to the cell membrane is necessary for cell transformation to occur (19). Although various forms of GAP have been isolated (20), the molecular mass appears to be about 120 kDa. Thus, none of the labeled proteins observed in the present study represent the GAPp21 complex. There are several possible reasons why we did not detect this complex. It is possible that the precipitating antibody, Y13-259, and GAP may bind to the same region of the p21 protein (5). Thus, the immunoprecipitated protein would not have GAP bound to it. Alternatively, the binding of GAP to the p21 protein might occur at a later time than we examined in our studies-i.e., 10 hr after the introduction of the p21 protein into these cells. Finally, it is possible that the SANPAH label used in the present studies is not present on residues of the p21 protein that interact with GAP or that it interferes with the association of p21 with GAP.


Further studies are required to reveal the structure and functional role of the putative 43-kDa protein that makes up, together with p21, the 64-kDA complex described in the present studies. Since this protein complex is quite specific and appears within a few hours after the introduction of p21 into cells, it may play a critical role in mediating the steps by which an activated RAS oncogene enhances the conversion of normal cells to tumor cells. This work was supported by a grant from the National Cancer Institute (CA42500) to M.R.P., a grant from the National Cancer Institute (CA02111) to I.B.W., and an award from the National Foundation for Cancer Research to I.B.W. 1. Tabin, C. J., Bradley, S. M., Bargmann, C. I., Weinberg, R. A., Papageorge, A. G., Scolnick, E. M., Dhar, R. & Chang, E. H. (1982) Nature (London) 300, 143-149. 2. Reddy, E. P., Reynolds, R. K., Santos, E. & Barbacid, M. (1982) Nature (London) 300, 149-153. 3. Fasano, O., Aldrich, T., Tamanoi, F., Taparowsky, E., Furth, M. E. & Wigler, M. (1984) Proc. Natl. Acad. Sci. USA 81, 4008-4012. 4. Gibbs, J., Sigal, I. S., Poe, M. & Scolnick, E. (1984) Nature (London) 310, 644-649. 5. Adari, H., Lowy, D. R., Willumsen, B. M., Der, C. J. & McCormick, F. (1988) Science 240, 518-521. 6. Vogel, U. S., Dixon, R. A. F., Shaber, M. D., Diehl, R. E., Marshall, M. S., Scolnick, E. M., Sigal, 1. S. & Gibbs, J. B. (1986) Nature (London) 335, 90-93. 7. de Gunzburg, J., Riehl, R. & Weinberg, R. A. (1989) Proc. Natl. Acad. Sci. USA 86, 4007-4011. 8. Wakelam, M. J. O., Davies, S. A., Houslay, M. D., McKay, I., Marshall, C. J. & Hall, A. (1986) Nature (London) 323, 173-176. 9. Lacal, J. C., Fleming, T. P., Warren, B. S., Blumberg, P. M. & Aaronson, S. A. (1987) Mol. Cell. Biol. 7, 4146-4149. 10. Backer, J. M. & Weinstein, 1. B. (1986) Proc. Natl. Acad. Sci. USA 83, 6357-6361. 11. Smith, M. R., DeGucidibus, S. J. & Stacey, D. W. (1986) Nature (London) 320, 540-543. 12. Ballmer-Hofer, K., Schlup, V., Burn, P. & Burger, M. M. (1982) Anal. Biochem. 126, 246-250. 13. Ronai, Z. & Weinstein, I. B. (1988) J. Virol. 62, 1057-1060. 14. Inoue, Y., Nakamori, H., Iwai, S., Ohtsuka, E., Ikehara, M., Miura, K., Noguchi, S. & Nishimura, S. (1986) Jpn. J. Cancer Res. (Gann) 77, 45-51. 15. Furth, M. E., Davis, L. J., Fleurdelys, B. & Scolnick, E. M. (1982) J. Virol. 43, 294-304. 16. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88. 17. Bar-Sagi, D. & Feramisco, J. R. (1986) Science 233, 1061-1068. 18. Stacey, D. W. & Kung, H.-F. (1984) Nature (London) 310, 508-511. 19. Willumsen, B. M., Papageorge, A. G., Hubbert, N. L., Beckesi, E., Kung, H.-F. & Lowy, D. R. (1984) EMBO J. 4, 2893-2896. 20. McCormick, F. (1989) Cell 56, 5-8.