Title: The Ras-related Protein Rheb Is Farnesylated and Antagonizes Ras Signaling and Transformation
Abstract: Presently, nothing is known about the function of the Ras-related protein Rheb. Since Rheb shares significant sequence identity with the core effector domains of Ras and KRev-1/Rap1A, it may share functional similarities with these two structurally related, yet functionally distinct, small GTPases. Furthermore, since like Ras, Rheb terminates with a COOH terminus that is likely to signal for farnesylation, it may be a target for the farnesyltransferase inhibitors that block Ras processing and function. To compare Rheb function with those of Ras and KRev-1, we introduced mutations into Rheb that generate constitutively active or dominant negative forms of Ras and Ras-related proteins and were designated Rheb(64L) and Rheb(20N), respectively. Expression of wild type or mutant Rheb did not alter the morphology or growth properties of NIH 3T3 cells. Thus, aberrant Rheb function is distinct from that of Ras and fails to cause cellular transformation. Instead, similar to KRev-1, co-expression of Rheb antagonized oncogenic Ras transformation and signaling. In vitro and in vivo analyses showed that like Ras, Rheb proteins are farnesylated and are sensitive to farnesyltransferase inhibition. Thus, it is possible that Rheb function may be inhibited by farnesyltransferase inhibitors treatment and, consequently, may contribute to the ability of these inhibitors to impair Ras transformation. Presently, nothing is known about the function of the Ras-related protein Rheb. Since Rheb shares significant sequence identity with the core effector domains of Ras and KRev-1/Rap1A, it may share functional similarities with these two structurally related, yet functionally distinct, small GTPases. Furthermore, since like Ras, Rheb terminates with a COOH terminus that is likely to signal for farnesylation, it may be a target for the farnesyltransferase inhibitors that block Ras processing and function. To compare Rheb function with those of Ras and KRev-1, we introduced mutations into Rheb that generate constitutively active or dominant negative forms of Ras and Ras-related proteins and were designated Rheb(64L) and Rheb(20N), respectively. Expression of wild type or mutant Rheb did not alter the morphology or growth properties of NIH 3T3 cells. Thus, aberrant Rheb function is distinct from that of Ras and fails to cause cellular transformation. Instead, similar to KRev-1, co-expression of Rheb antagonized oncogenic Ras transformation and signaling. In vitro and in vivo analyses showed that like Ras, Rheb proteins are farnesylated and are sensitive to farnesyltransferase inhibition. Thus, it is possible that Rheb function may be inhibited by farnesyltransferase inhibitors treatment and, consequently, may contribute to the ability of these inhibitors to impair Ras transformation. Mutated forms of the three ras genes (H-, K-, and N-ras) are associated with 30% of all human cancers and encode potent transforming and oncogenic mutant proteins (1Clark G.J. Der C.J. Dickey B.F. Birnbaumer L. GTPases in Biology I. Springer-Verlag, Berlin1993: 259-288Google Scholar). Normal Ras proteins function as GDP/GTP-regulated molecular switches (2Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar). Guanine nucleotide exchange factors (SOS and RasGRF/CDC25) promote formation of the active, GTP-bound state (2Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar, 3Quilliam L.A. Khosravi-Far R. Huff S.Y. Der C.J. BioEssays. 1995; 17: 395-404Crossref PubMed Scopus (193) Google Scholar, 4Feig L.A. 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Consequently, mutated Ras proteins cause constitutive, ligand-independent activation of these pathways, thereby promoting to the aberrant growth of tumor cells. Ras proteins are prototypes for a large superfamily of Ras-related proteins (>60 mammalian members) that function as GDP/GTP-regulated molecular switches (2Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar, 6Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 349: 117-127Crossref Scopus (2698) Google Scholar, 9Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1844) Google Scholar). However, despite their strong amino acid sequence identity with Ras proteins (30–55%), the majority of these small GTPases lack the potent transforming potential of Ras proteins. Exceptions include TC21/R-Ras2 (10Chan A.M.L. Miki T. Meyers K.A. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7558-7562Crossref PubMed Scopus (86) Google Scholar, 11Graham S.M. 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Sci. 1993; 18: 250-254Abstract Full Text PDF PubMed Scopus (194) Google Scholar). However, although the Raf-1 serine/threonine kinase is clearly a critical effector important for Ras signaling and transformation (22Kolch W. Heidecker G. Lloyd P. Rapp U.R. Nature. 1991; 349: 426-428Crossref PubMed Scopus (355) Google Scholar, 23Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar, 24Pagès G. Lenormand P. L'Allemain G. Chambard J.-C. Meloche S. Pouysségur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8319-8323Crossref PubMed Scopus (926) Google Scholar, 25Westwick J.K. Cox A.D. Der C.J. Cobb M.H. Hibi M. Karin M. Brenner D.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6030-6034Crossref PubMed Scopus (168) Google Scholar, 26Troppmair J. Bruder J.T. Munoz H. Lloyd P.A. Kyriakis J. Banerjee P. Avruch J. Rapp U.R. J. Biol. Chem. 1994; 269: 7030-7035Abstract Full Text PDF PubMed Google Scholar, 27Brtva T.R. Drugan J.K. Ghosh S. Terrell R.S. Campbell-Burk S. Bell R.M. Der C.J. J. Biol. Chem. 1995; 270: 9809-9812Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), neither TC21 nor R-Ras cause activation of Raf-1 (28Graham S.M. Vojtek A.B. Huff S.Y. Cox A.D. Clark G.J. Cooper J.A. Der C.J. Mol. Cell. Biol. 1996; 16: 6132-6140Crossref PubMed Scopus (54) Google Scholar, 29Huff S.Y. Quilliam L.A. Cox A.D. Der C.J. Oncogene. 1997; 14: 133-143Crossref PubMed Scopus (43) Google Scholar). Thus, despite possessing complete identity with the core Ras effector domain, these two Ras-related proteins must cause transformation by activation of Raf-independent effector pathways. Therefore, while the core effector domain sequences of Ras, TC21, and R-Ras are clearly critical for effector interactions, sequences flanking this core region are likely to influence specific effector interactions. Consistent with this, recent mutagenesis studies have extended the boundaries of the Ras effector domain to include Ras residues 25–45 (30Zhang K. Noda M. Vass W.C. Papageorge A.G. Lowy D.R. Science. 1990; 249: 162-165Crossref PubMed Scopus (81) Google Scholar, 31Akasaka K. Tamada M. Wang F. Kariya K. Shima F. Kikuchi A. Yamamoto M. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1996; 271: 5353-5360Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Although KRev-1/Rap1A also shares complete identity with the core Ras effector domain, it lacks any transforming potential and, instead, antagonizes the ability of Ras to transform cells (32Kitayama H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Abstract Full Text PDF PubMed Scopus (763) Google Scholar, 33Kitayama H. Matsuzaki T. Ikawa Y. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4284-4288Crossref PubMed Scopus (104) Google Scholar, 34Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (335) Google Scholar). Since KRev-1 can interact with the Raf-1 serine/threonine kinase (35Zhang X. Settleman J. Kyriakis J.M. Takeuchi-Suzuki E. Elledge S.J. Marshall M.S. Bruder J.T. Rapp U.R. Avruch J. Nature. 1993; 364: 308-313Crossref PubMed Scopus (689) Google Scholar), as well as other candidate Ras effectors (e.g. RalGDS and related proteins) (36Spaargaren M. Bischoff J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12609-12613Crossref PubMed Scopus (249) Google Scholar, 37Peterson S.N. Trabalzini L. Brtva T.R. Fischer T. Altschuler D.L. Martelli P. Lapetina E.G. Der C.J. White G.C., II J. Biol. Chem. 1996; 271: 29903-29908Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), it is not clearly understood why KRev-1 is not transforming and how it antagonizes Ras function. One possibility is that KRev-1 interacts with, but fails to activate, Ras effectors important for Ras transformation. Thus, KRev-1 may form nonproductive complexes with Raf and other Ras effectors, thereby preventing their association with Ras. Consistent with this possibility KRev-1 fails to activate Raf or other downstream signaling activities that are stimulated by Ras. Rheb (Ras homolog highly enriched in brain) is a recently identified member of the Ras superfamily (38Yamagata K. Sanders L.K. Kaufmann W.E. Yee W. Barnes C.A. Nathans D. Worley P.F. J. Biol. Chem. 1994; 269: 16333-16339Abstract Full Text PDF PubMed Google Scholar). The overall sequence of Rheb is distinct from other Ras-related proteins, but it shares strongest overall amino acid homology with human Rap2, yeast RAS1, and human H-Ras. Rheb also possesses sequence characteristics in common with Ras and Rap proteins. First, Rheb shows strong identity with the Ras and Rap core effector domains, suggesting that Rheb may share common effector interactions with Ras and KRev-1. The equivalent region of Rheb is quite similar to Ras with the first six amino acids being identical. Second, Rheb terminates with a CAAX motif (C, cysteine, A, aliphatic, andX, terminal amino acid) that is likely to signal for posttranslational modification by the C15 farnesyl isoprenoid (39Maltese W.A. FASEB J. 1990; 4: 3319-3328Crossref PubMed Scopus (430) Google Scholar, 40Cox A.D. Der C.J. Curr. Opin. Cell Biol. 1992; 4: 1008-1016Crossref PubMed Scopus (201) Google Scholar, 41Magee A.I. Newman C.M.H. Giannakouros T. Hancock J.F. Fawell E. Armstrong J. Biochem. Soc. Trans. 1992; 20: 497-499Crossref PubMed Scopus (34) Google Scholar). Except for the Ras proteins, Rap2B and two Rho family proteins (RhoB and RhoE) (42Adamson P. Marshall C.J. Hall A. Tilbrook P.A. J. Biol. Chem. 1992; 267: 20033-20038Abstract Full Text PDF PubMed Google Scholar, 43Farrell F.X. Yamamoto K. Lapetina E.G. Biochem. J. 1993; 289: 349-355Crossref PubMed Scopus (39) Google Scholar, 44Foster R. Hu K.-Q. Lu Y. Nolan K.M. Thissen J. Settleman J. Mol. Cell. Biol. 1996; 16: 2689-2699Crossref PubMed Scopus (241) Google Scholar), all other prenylated Ras superfamily proteins are modified by the related C20geranylgeranyl isoprenoid. Hence, Rheb function may be antagonized by FTIs that block Ras function (45Sattler I. Tamanoi F. Maruta H. Regulation of the RAS Signaling Network. R. G. Landes, Austin, TX1996Google Scholar). In the present study, we introduced putative gain or loss of function mutations into Rheb and evaluated their biological properties in NIH 3T3 cells. We found that overexpression of wild type or mutant Rheb failed to cause morphologic or growth transformation of NIH 3T3 cells. Instead, like KRev-1, we observed that Rheb co-expression inhibited oncogenic Ras signaling and transformation and that Rheb complexed with Raf-1 in vitro. However, whereas KRev-1 is modified by the geranylgeranyl isoprenoid, like Ras, we determined that Rheb is modified by farnesylation, and this processing is blocked by FTI treatment. Finally, Rheb displays a subcellular location that is similar to that of Ras rather than the intracellular membrane location seen with KRev-1. Taken together, these results suggest that Rheb function is similar to that of KRev-1 rather than Ras and that FTI inhibition of Rheb function may be a component of FTI inhibition of Ras transformation. We utilized polymerase chain reaction-mediated DNA amplification to isolate sequences corresponding to rheb from a randomly primed rat fetal liver cDNA library (generous gift of T. Dawson). Five μl (108 plaque-forming units/ml) of the library was subjected to 30 cycles of amplification at an annealing temperature of 55 °C using 5′- and 3′-oligonucleotides that contain the coding sequences for the NH2- or COOH-terminal rat Rheb protein sequence (38Yamagata K. Sanders L.K. Kaufmann W.E. Yee W. Barnes C.A. Nathans D. Worley P.F. J. Biol. Chem. 1994; 269: 16333-16339Abstract Full Text PDF PubMed Google Scholar), together with flanking noncoding sequences that containBamHI restriction sites to facilitate subcloning into theBamHI site of the Bluescript SKII plasmid vector. The fidelity of the sequence was confirmed by dideoxy sequencing. Therheb cDNA fragment was then subcloned into theBamHI site of the pZIP-NeoSV(x)1 retrovirus for expression from the Moloney long terminal repeat promoter or the pCGN-hyg mammalian expression vector for expression of an NH2-terminal hemagglutinin (HA) 1The abbreviations used are: HA, hemagglutinin; MAPK, mitogen-activated protein kinase; FPP, farnesylpyrophosphate; GGPP, geranylgeranylpyrophosphate; FTI, farnesyltransferase inhibitor; GMPPCP, guanylyl β,γ-methylenediphosphonate; CAT, chloramphenicol acetyltransferase. epitope-tagged fusion protein (HA-Rheb) from the cytomegalovirus promoter (46Cepko C.L. Roberts B. Mulligan R.C. Cell. 1984; 37: 1053-1062Abstract Full Text PDF PubMed Scopus (641) Google Scholar, 47Tanaka M. Herr W. Cell. 1990; 60: 375-386Abstract Full Text PDF PubMed Scopus (517) Google Scholar). Therheb cDNA sequence was also subcloned into the pGEX-2T bacterial expression vector to encode a glutathioneS-transferase (GST)-Rheb chimeric fusion protein. Oligonucleotide-directed mutagenesis (Stratagene Chameleon Mutagenesis kit) was used to generate mutant rheb or Krev-1 cDNA sequences. All mutated sequences were sequenced to confirm fidelity. NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and plasmid DNA transfections were performed by calcium phosphate precipitation using procedures that we have described previously (48Clark G.J. Cox A.D. Graham S.M. Der C.J. Methods Enzymol. 1995; 255: 395-412Crossref PubMed Scopus (182) Google Scholar). Focus formation inhibition assays were performed by co-transfecting NIH 3T3 cells in triplicate with oncogenic H-Ras(12V) driven by its genomic promoter (pUC-rasH(12V)) and a 10-fold molar excess of each rheb expression construct. The number of transformed foci was determined after 16 days. To determine the consequence of exogenously introduced wild type or mutant Rheb protein expression on the growth of NIH 3T3 cells, cells stably transfected with expression vectors encoding wild type or mutant Rheb proteins were isolated by selection of transfected cells in growth medium supplemented with 500 μg/ml G418 (Geneticin; Life Technologies, Inc.). Multiple, G418-resistant colonies (>100) were pooled to establish cell lines stably transfected with each rhebexpression vector and were then used for in vitro growth assays (on plastic or in soft agar) using procedures described previously (48Clark G.J. Cox A.D. Graham S.M. Der C.J. Methods Enzymol. 1995; 255: 395-412Crossref PubMed Scopus (182) Google Scholar). Growth curves were performed by plating cells at 4 × 104 cells per 60-mm dish. Transient transfection transcription assays were done to determine if Rheb can activate transcription or block oncogenic Ras stimulation of transcription, from the ets/AP-1 Ras-responsive promoter element using procedures described previously (49Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. Van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Crossref PubMed Scopus (330) Google Scholar). Briefly, cultures of NIH 3T3 cells were transfected with the pBL4X-CAT reporter plasmid, where chloramphenicol acetyltransferase (CAT) gene expression is regulated by an ets/AP-1 containing promoter and the indicated Rheb and/or Ras expression plasmids. After 48 h the cells were lysed and assayed for CAT activity as described previously (49Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. Van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Crossref PubMed Scopus (330) Google Scholar). To generate Rheb-specific antiserum, we utilized Rheb COOH-terminal sequences that are distinct from the sequences of corresponding COOH-terminal sequences of Ras, Rap, and other Ras-related proteins (38Yamagata K. Sanders L.K. Kaufmann W.E. Yee W. Barnes C.A. Nathans D. Worley P.F. J. Biol. Chem. 1994; 269: 16333-16339Abstract Full Text PDF PubMed Google Scholar). Polymerase chain reaction-mediated DNA amplification was used to introduce sequences corresponding to the Rheb COOH-terminal 33 amino acid residues (designated Rheb/C) into the pGEX-2T bacterial expression vector for expression of the GST·Rheb/C fusion protein. Purified GST·Rheb/C protein was injected intramuscularly into rabbits, followed by a second injection after 6 weeks. Antiserum with reactivity against Rheb was used to isolate affinity purified anti-Rheb antibodies using GST·Rheb/C, and the resulting affinity purified anti-Rheb antibodies were used for Western blot analysis of Rheb protein expression. To determine if Rheb could bind to activated human Raf-1, Sf9 insect cells were co-infected with recombinant baculovirus expressing full-length Raf-1 and activated Ras (generous gift of J. Strom), then lysed in Nonidet P-40-containing detergent lysis buffer, and clarified by centrifugation at 103 rpm. GST-fusion proteins containing Ras, KRev-1, and Rheb sequences were first preloaded with nonhydrolyzable GTP (GMPPCP) and then incubated with 10 μl of the Raf-1 containing insect cell lysate in phosphate-buffered saline, 10 mm dithiothreitol for 1 h at 4 °C. The GST-fusion proteins were then isolated and resolved on SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to Immobilon membrane, probed with an anti-Raf antibody (C-12, Santa Cruz Biotech), and visualized by enhanced chemiluminescence. Oocyte lysate and activated Ras protein were prepared as described previously (50Shibuya E.K. Morris J. Rapp U.R. Ruderman J.V. Cell Growth & Differ. 1996; 7: 235-241PubMed Google Scholar). GST-Rheb and GST-KRev-1 fusion proteins were first preloaded with GMPPCP (10 μg), then added to the oocyte lysate together with 100 ng of GTP charged H-Ras, an ATP regeneration system, and incubated at 20 °C. Samples were taken at time 0 and at 2 h and then assayed for their ability to phosphorylate myelin basic protein as described previously (51Clark G.J. Drugan J.K. Terrell R.S. Bradham C. Der C.J. Bell R.M. Campbell-Burk S. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 1577-1581Crossref Scopus (66) Google Scholar). For in vitroprenylation analysis (52Khosravi-Far R. Der C.J. Methods Enzymol. 1995; 255: 46-60Crossref PubMed Scopus (12) Google Scholar), 5 μg of recombinant H-Ras, GST-KRev-1, and GST-Rheb proteins were added to 45 μl of rabbit reticulocyte lysate (Promega), together with [3H]mevalonate, [3H]-farnesylpyrophosphate (FPP), or [3H]geranylgeranylpyrophosphate (GGPP) (DuPont NEN) (25, 2.5, and 2.5 μCi, respectively), either with or without the indicated concentrations of the FTI-277 FTI. We previously demonstrated that FTI-277 can inhibit potently farnesyltransferase in vitro(IC50 = 500 pm), is highly selective for farnesyltransferase over geranylgeranyltransferase I (IC50= 50 mm), and can selectively inhibit prenylation of H-Ras, but not geranylgeranylated KRev-1, in whole cells (53Lerner E.C. Qian Y. Blaskovich M.A. Fossum R.D. Vogt A. Sun J. Cox A.D. Der C.J. Hamilton A.D. Sebti S.M. J. Biol. Chem. 1995; 270: 26802-26806Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). For prenylation analysis in NIH 3T3 cells, cells stably expressing wild type Ras or Rheb were incubated with varying concentrations of FTI-277 for 72 h. The cells were then fractionated into crude membrane (P100) and supernatant fractions (S100) as described previously (54Cox A.D. Solski P.A. Jordan J.D. Der C.J. Methods Enzymol. 1995; 255: 195-221Crossref PubMed Scopus (39) Google Scholar), then resolved by SDS-polyacrylamide gel electrophoresis, and exposed for autoradiography to visualize incorporated label. Cells expressing HA epitope-tagged H-Ras, Rheb, or KRev-1 protein were grown on coverslips for 18 h at 37 °C and then fixed with 3.7% formaldehyde in phosphate-buffered saline and permeabilized with 0.5% Triton X-100 in Tris-buffered saline (150 mm NaCl, 50 mm Tris, pH 7.6, 0.1% NaN3). To determine the localization of the tagged proteins, anti-HA monoclonal antibody (Babco) was diluted 1:50 and used to stain the fixed cells for 1 h at 25 °C. The cells were then washed in phosphate-buffered saline and incubated with rhodamine-conjugated goat anti-mouse antibody (Cappel, Durham, NC) for 1 h at 25 °C. Coverslips were viewed on a Zeiss Axiophot microscope, and fluorescence micrographs were taken on T-max 400 film (Eastman Kodak Co.). Rheb shows strong sequence homology with the Ras effector domain (Fig. 1). To determine whether Rheb function could regulate cell growth, we generated mutant Rheb proteins with mutations analogous to those that convert Ras and other Ras-related proteins into constitutively active or inactive proteins. Whereas Rheb(64L) contains a mutation that is analogous to the Q61L mutation that renders Ras proteins constitutively active and transforming (1Clark G.J. Der C.J. Dickey B.F. Birnbaumer L. GTPases in Biology I. Springer-Verlag, Berlin1993: 259-288Google Scholar), Rheb(20N) contains a mutation that is analogous to the S17N mutation that results in dominant negative mutants of Ras and Ras-related proteins (28Graham S.M. Vojtek A.B. Huff S.Y. Cox A.D. Clark G.J. Cooper J.A. Der C.J. Mol. Cell. Biol. 1996; 16: 6132-6140Crossref PubMed Scopus (54) Google Scholar, 55Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar, 56Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3084) Google Scholar, 57Zhang Z. Vuori K. Wang H.-G. Reed J.C. Ruoslahti E. Cell. 1996; 85: 61-69Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). To evaluate the consequences of Rheb protein expression on the growth properties of NIH 3T3 cells in vitro, we isolated NIH 3T3 cells stably transfected with expression vectors that encoded wild type or mutant Rheb proteins. To avoid clonal variation differences that may complicate our comparisons, we utilized early passages of pooled populations of multiple G418-resistant colonies (>100) for our analyses. We first determined if these G418-resistant cell populations expressed the exogenously introduced Rheb sequences. Western blot analysis using a rabbit anti-Rheb polyclonal antiserum that recognized the unique COOH-terminal amino acid sequence of Rheb was done. Consistent with the divergence of this portion of Rheb with other Ras-related proteins (58Valencia A. Chardin P. Wittinghofer A. Sander C. Biochemistry. 1991; 30: 4637-4648Crossref PubMed Scopus (56) Google Scholar), this antiserum did not recognize the closely related H-Ras, KRev-1, or TC21/R-Ras2 proteins (data not shown). A 21-kDa protein was detected in the control, empty vector-transfected cells, indicating that Rheb is expressed in NIH 3T3 cells (Fig.2 A). The Rheb(WT)-transfected cells expressed a 2- to 3-fold elevation of protein at this position, whereas a slower migrating band was detected in the Rheb(64L)-transfected cells. This altered mobility is seen when the equivalent mutation is introduced into Ras and Ras-related proteins. Cells transfected with Rheb(20N) did not show elevated nor altered forms of protein. This is similar to observations with 17N mutant versions of Ras and Ras-related proteins, where expression is typically lower than that seen with the wild type protein (29Huff S.Y. Quilliam L.A. Cox A.D. Der C.J. Oncogene. 1997; 14: 133-143Crossref PubMed Scopus (43) Google Scholar, 55Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar). This may be due to a reduced stability of the protein or, alternatively, to the fact that high level expression of this mutant is not tolerated by NIH 3T3 cells. We and others (48Clark G.J. Cox A.D. Graham S.M. Der C.J. Methods Enzymol. 1995; 255: 395-412Crossref PubMed Scopus (182) Google Scholar) have shown previously that constitutively activated mutants of Ras cause both morphologic and growth transformation of NIH 3T3 cells. In contrast, we found that cells transfected with wild type or mutant rheb sequences showed morphologies that were indistinguishable from that of untransformed NIH 3T3 cells (data not shown). Additionally, in contrast to observations with pZIP-ras(17N)-transfected NIH 3T3 cells (55Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar), no significant reduction in the appearance of G418-resistant colonies was seen for pZIP-rheb(20N)-transfected cells (data not shown). We then determined if the growth rates of NIH 3T3 cells were altered by expression of exogenously introduced rheb sequences. As shown in Fig. 2 B, the growth rates on plastic of wild type or 64L rheb-transfected cell populations were indistinguishable from those displayed by the empty vector-transfected cells. Furthermore, whereas Ras-transformed NIH 3T3 cells will proliferate in growth medium supplemented with low serum (1–5%) (data not shown) (48Clark G.J. Cox A.D. Graham S.M. Der C.J. Methods Enzymol. 1995; 255: 395-412Crossref PubMed Scopus (182) Google Scholar), all three Rheb-transfected populations showed a requirement for growth medium supplemented with 10% calf serum (Fig.2 C). Finally, we also tested their ability to form colonies in soft agar and found them to be negative (data not shown). Thus, in contrast to Ras, aberrant wild type or mutant Rheb function did not cause growth transformation, or inhibition, of NIH 3T3 cells. Since Rheb lacked the growth promoting activity seen with constitutively activated mutants of Ras, TC21, or R-Ras, we next determined if Rheb was more similar to KRev-1/Rap1A and, instead, could antagonize Ras function (32Kitayama H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Abstract Full Text PDF PubMed Scopus (763) Google Scholar, 33Kitayama H. Matsuzaki T. Ikawa Y. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4284-4288Crossref PubMed Scopus (104) Google Scholar). For these analyses, we used the constitutively activated KRev-1(63E) mutant as a positive control for these co-transfection focus formation inhibition assays (33Kitayama H. Matsuzaki T. Ikawa Y. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4284-4288Crossref PubMed Scopus (104) Google Scholar). Like the consequences of co-transfection of pZIP-Krev-1(63E), co-transfection of pZIP expression vectors encoding Rheb(WT) or Rheb(64L) also caused a greater than 50% reduction in oncogenic H-Ras(12V) focus-forming activity (Fig. 3 A). In contrast, co-transfection with Rheb(20N) did not cause a