K- and N-Ras Are Geranylgeranylated in Cells Treated with Farnesyl Protein Transferase Inhibitors

The association of mutant forms of Ras protein with a variety of human cancers has stimulated intense interest in therapies based on inhibiting oncogenic Ras signaling. Attachment of Ras proteins to the plasma membrane is required for effective Ras signaling and is initiated by the enzyme farnesyl protein transferase. We found that in the presence of potent farnesyl protein transferase inhibitors, Ras proteins in the human colon carcinoma cell line DLD-1 were alternatively prenylated by geranylgeranyl transferase-1. When H-Ras, N-Ras, K-Ras4A, and K-Ras4B were expressed individually in COS cells, H-Ras prenylation and membrane association were found to be uniquely sensitive to farnesyl transferase inhibitors; N- and K-Ras proteins incorporated the geranylgeranyl isoprene group and remained associated with the membrane fraction. The alternative prenylation of N- and K-Ras has significant implications for our understanding of the mechanism of action of farnesyl protein transferase inhibitors as anti-cancer chemotherapeutics. The association of mutant forms of Ras protein with a variety of human cancers has stimulated intense interest in therapies based on inhibiting oncogenic Ras signaling. Attachment of Ras proteins to the plasma membrane is required for effective Ras signaling and is initiated by the enzyme farnesyl protein transferase. We found that in the presence of potent farnesyl protein transferase inhibitors, Ras proteins in the human colon carcinoma cell line DLD-1 were alternatively prenylated by geranylgeranyl transferase-1. When H-Ras, N-Ras, K-Ras4A, and K-Ras4B were expressed individually in COS cells, H-Ras prenylation and membrane association were found to be uniquely sensitive to farnesyl transferase inhibitors; N- and K-Ras proteins incorporated the geranylgeranyl isoprene group and remained associated with the membrane fraction. The alternative prenylation of N- and K-Ras has significant implications for our understanding of the mechanism of action of farnesyl protein transferase inhibitors as anti-cancer chemotherapeutics. Newly synthesized Ras proteins are partitioned to the cytoplasmic face of the plasma membrane by a series of post-translational modifications. The first step, catalyzed by the enzyme farnesyl protein transferase, is the addition of the 15- carbon isoprenyl group farnesyl to the sulfhydryl group of cysteine in the Ras carboxyl-terminal CAAX box (where C is cysteine, A is aliphatic, andX is typically Met or Ser) (1Casey P.J. J. Lipid Res. 1992; 33: 1731-1740Abstract Full Text PDF PubMed Google Scholar, 2Reiss Y. Stradley S.J. Gierasch L.M. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 732-736Crossref PubMed Scopus (312) Google Scholar, 3Sinensky M. Lutz R.J. Bioessays. 1992; 14: 25-31Crossref PubMed Scopus (143) Google Scholar). Farnesylation is followed by proteolytic removal of the AAX amino acids and methylation of the carboxyl group of the farnesylated cysteine (4Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1746) Google Scholar). Ras proteins at the plasma membrane cycle between an active GTP-bound state and an inactive GDP-bound state. Mutations that stabilize the active GTP-bound state have been identified in over 30% of human tumors, with particularly high incidences in pancreatic (∼90%) and colon (∼50%) cancers. Four oncogenic Ras proteins have been described, H-Ras, N-Ras, K-Ras4A, and K-Ras4B. The majority of mutations associated with human cancer have been found in the K-Ras gene. The two K-Ras proteins are products of a single alternatively spliced transcript, with K-Ras4B the predominant isoform (>80%) (5Bos J.L. Mutat. Res. 1988; 195: 255-271Crossref PubMed Scopus (679) Google Scholar, 6Barbacid M. Annu. Rev. Biochem. 1987; : 779-827Crossref PubMed Scopus (3787) Google Scholar). Ras proteins that have been genetically modified so that they lack the isoprenylated cysteine do not associate with the plasma membrane and cannot transform fibroblasts (7Kato K. Cox A.D. Hisaka M.M. Grahm S.M. Buss J.E. Der C.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6403-6407Crossref PubMed Scopus (557) Google Scholar). These genetic experiments provided the basis for the development of farnesyl transferase inhibitors (FTIs) 1The abbreviations used areFTIfarnesyl protein transferase inhibitor;PCRpolymerase chain reaction;DMEMDulbecco's modified Eagle's medium;HPLChigh pressure liquid chromatography. as anti-cancer agents. A number of reports have demonstrated that pharmacological inhibition of farnesyl protein transferase by CAAX analogs reduces anchorage-independent growth of Ras-transformed cells in soft agar (8Sepp-Lorenzino L. Ma Z. Rands E. Kohl N.E. Gibbs J.B. Oliff A. Rosen N. Cancer Res. 1995; 55: 5302-5309PubMed Google Scholar) and slows growth of Ras-transformed cells in nude mice (9Kohl N.E. Mosser S.D. deSolms S.J. Giuliani E.A. Pompliano D.L. Graham S.L. Smith R.L. Scolnick E.M. Oliff A. Gibbs J.B. Science. 1993; 260: 1934-1937Crossref PubMed Scopus (620) Google Scholar, 10Kohl N.E. Wilson F.R. Mosser S.D. Giuliani E. DeSolms S.J. Conner M.W. Anthony N.J. Holtz W.J. Gomez R.P. Lee T.-J. Smith R.L. Grahm S.L. Hartman G.D. Gibbs J.B. Oliff A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9141-9145Crossref PubMed Scopus (324) Google Scholar). The FTIs appear relatively non-toxic in that they do not interfere with normal cell proliferation (11James G.L. Brown M.S. Cobb M.H. Goldstein J.L. J. Biol. Chem. 1994; 269: 27705-27714Abstract Full Text PDF PubMed Google Scholar). This result was somewhat surprising because Ras function was shown to be necessary for normal growth factor signaling and cell proliferation (12Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1127) Google Scholar). A mechanism through which cells may proliferate in the presence of FTIs was suggested by the observation that, in vitro, the K-Ras4B protein, but not H-Ras, can act as a substrate for geranylgeranyl transferase-1 (13James G.L. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 6221-6226Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Geranylgeranyl transferase-1 adds the 20 carbon geranylgeranyl isoprenyl unit to the cysteine residue of the CAAX motif (14Casey P.J. Thissen J.A. Moomaw J.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8631-8635Crossref PubMed Scopus (158) Google Scholar). Geranylgeranyl transferase-1 substrate proteins generally have a carboxyl-terminal leucine (15Kinsella B.T. Erdman R.A. Maltese W.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8934-8938Crossref PubMed Scopus (72) Google Scholar). James et al. (16James G. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4454-4458Crossref PubMed Scopus (139) Google Scholar) recently reported that a chimeric Ras protein consisting of the first 164 amino acids of H-RasV12 followed by the carboxyl-terminal 24 amino acids of K-Ras4B incorporates 3Hmevalonate in Rat-1 cells even in the presence of the peptidomimetic FTIs. The prenyl group attached to H/K-Ras4BV12 in this study was reported to be farnesyl, both in the absence and presence of the FTI. We report here that Ras proteins in the human cancer cell line DLD-1 become geranylgeranylated in the presence of farnesyl transferase inhibitors. Plasmids expressing H-, N-, and the two K-Ras isoforms were transfected into COS cells to determine the effects of FTI on the prenylation and membrane association of each isoform. farnesyl protein transferase inhibitor; polymerase chain reaction; Dulbecco's modified Eagle's medium; high pressure liquid chromatography. 3Hmevalonolactone (40 Ci/mmol) in 100% ethanol at 1 mCi/ml was from DuPont NEN (NET-1075). It was dried under nitrogen to increase the concentration to 100 mCi/ml. G418 and trypsin-EDTA were from Life Technologies, Inc.; Amplify fluorographic reagent was from Amersham Life Science; Pefabloc SC was from Boehringer Mannheim. All other chemicals were from Sigma. The pMev construct, which contains the coding sequence for the mevalonate transporter under control of a cytomegalovirus immediate early gene promoter (17Kim C.M. Goldstein J.L. Brown M.S. J. Biol. Chem. 1992; 267: 23113-23121Abstract Full Text PDF PubMed Google Scholar) was co-transfected with a neo-expressing plasmid (pCI-neo, Promega Corp.) into DLD-1 cells (American Type Culture Collection, Rockville, MD) using Lipofectamine as described by the manufacturer (Life Technologies, Inc.). Control cells were transfected with pCI-neo alone. Neo-expressing colonies were selected in DMEM, 10% fetal calf serum containing 800 μm G418 and maintained in 400 μm G418. Treatment of the parental line, control neo-resistant lines, and pMev transfectants with mevastatin alone (20 μm) resulted in complete cell killing within several days. In the presence of exogenous mevalonate (100 μm), several pMev-transfected clones were resistant to 20 μmmevastatin. In contrast, pCI-neo transfectants and the parental line were equally sensitive to mevastatin in the presence or absence of exogenous mevalonate. DLDpMev cells were incubated overnight (18 h) at 37 °C, 5% CO2with SCH 44342 or 56582 at 0, 1.25, 2.5, 5.0, 10, and 20 μm, or with the farnesyl pyrophosphate antagonist, α-hydroxyfarnesyl-phosphonic acid (Biomol Research Laboratories, Inc., Plymouth Meeting, PA), at 0, 0.08, 0.2, 0.7, 2.2, and 6.6 μm. The following morning, the cells were treated with fresh compound or vehicle control, mevastatin (20 μm, to block endogenous mevalonate synthesis), and 3Hmevalonolactone (40 Ci/mmol) at 100 μCi/ml. After 18 h of incubation, the cells were rinsed twice with phosphate-buffered saline and lysed in RIPA buffer (150 mmNaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mm Tris, pH 7.5, 50 μm leupeptin, 1 mm Pefabloc SC, 2 μg/ml aprotinin, 2 μg/ml soybean trypsin inhibitor, 1 μg/ml pepstatin, 2 mm benzamide, 2 mm EDTA). Ras proteins were immunoprecipitated from approximately 20 million cpm of lysate in a 1-ml volume of RIPA buffer. Antibodies used were the pan-Ras antibody, Y13–259 (SC-35-agarose conjugate), or the K-Ras- (SC-30) or N-Ras- (SC-31) specific antibodies from Santa Cruz Biotechnology, Inc. To precipitate with Y13–259, 30 μl of agarose conjugate at 2 μg/μl was added to the lysate and rotated at 4 °C for 4 h. To precipitate with free antibody, 50 μl of SC-30 or SC-31 at 0.1 μg/μl was added and rotated at 4 °C for 3 h, and then 30 μl of protein A-agarose (Oncogene Science) resuspended in RIPA was added for 1 h with continued rotation. All prenyl analysis was done with Y13–259 immunoprecipitated Ras since SC-30 and SC-31 yielded insufficient labeled Ras for analysis. Total lysate (approximately 2 × 106 cpm) and immunoprecipitated Ras (100% of immunoprecipitate) were loaded on 18 × 18 cm 14% polyacrylamide gels and run at 30 mA for approximately 4 h. The gel was fixed in 25% methanol, 7% acetic acid for 15 min, immersed for 15 min in Amplify fluorographic reagent, dried, and exposed (Hyperfilm-MP, Amersham Life Science) for 1–30 days. Mutant (G12V) H-Ras cDNA was PCR amplified from human bladder carcinoma cell line T24 cDNA and cloned into the mammalian expression vector pSV-Sport (Life Technologies, Inc.). Mutant (G12V) K-Ras4B cDNA was PCR amplified from the human colon cancer line SW620 and cloned into the mammalian expression vector pCI-neo (Promega Corp.). Mutant (Q61H) K-Ras4A was PCR amplified from HTB-177 cells and cloned into pCI-neo. Mutant (G12N) N-Ras coding sequence, a gift from A. Wolfman (The Cleveland Clinic, Cleveland, OH), was cloned into the pCI-neo vector. All PCR products were sequenced and were transforming in NIH 3T3 cells. COS-7 cells were co-transfected (Lipofectamine, Life Technologies, Inc.) with 15 μg each of pMEV and H-, N-, K-Ras4A, or K-Ras4B expression vector and incubated for 48 h in control DMEM medium or DMEM with 7.5 μm SCH-56582. For the final 18 h, cells were treated with 3Hmevalonolactone (40 Ci/mmol) at 100 μCi/ml and 20 μm mevastatin. The cells were lysed in RIPA buffer, and Ras proteins were immunoprecipitated with Y13–259-agarose conjugate (Santa Cruz Biotechnology, Inc.). Structural characterization of prenyl groups attached to immunoprecipitated proteins was performed as described (18Casey P.J. Solski P.A. Der C.J. Buss J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8323-8327Crossref PubMed Scopus (782) Google Scholar). Briefly, protein was precipitated in acetone, washed twice with acetone to remove free lipids, and digested with trypsin overnight at 37 °C in 100 mm Tris-Cl, pH 7.5, 5% acetonitrile, and .04% w/v trypsin-EDTA. Cleavage of prenyl groups was initiated by addition of 0.1 volumes of methyl iodide in 3% formic acid. Released prenyl groups were extracted into chloroform:methanol (9:1), dried under nitrogen, dissolved in 100 μl of 50% acetonitrile, 25 mm phosphoric acid, and resolved on a C18 HPLC column (Waters 04698). COS-7 cells were transfected with mutant H-, N-, K-4A, K-4B, or H-Ras-CVLL expression vectors or vector control (pCI-neo) in the presence of 7.5 μm SCH 56582, 20 μmmevastatin (Sigma), or vehicle. Following transfection, cells were suspended in 0.9 ml of ice-cold hypotonic buffer (25 mmTris-HCl, pH 8.0, 1 mm EDTA, 5 μg/ml leupeptin, 1 mm Pefabloc SC, 50 μg/ml aprotinin, 5 μg/ml soybean trypsin inhibitor, 4 mm benzamidine) and sonicated for 5 s. The cell debris was pelleted at 1500 × g for 4 min at 4 °C and discarded, and the supernatant was transferred to Beckman polyallomer tubes and spun at 46,000 rpm (100,000 ×g) for 45 min. The pellet was washed once with ice-cold hypotonic buffer with protease inhibitors, re-pelleted at 46,000 rpm for 15 min, and then resuspended in 200 μl of ice-cold hypotonic buffer with protease inhibitors. The supernatant was concentrated in Microcon 10 concentrators (Amicon). Particulate and soluble (approximately of were loaded on an 18 × 18 cm 14% polyacrylamide run at 30 mA for approximately 4 h, and then transferred to Corp.). The was for 30 min in 5% dried in (20 mm pH mm and incubated with for for H-Ras, SC-31 for N-Ras (Santa at 1 μg/ml in 0.5% fetal calf serum for 1 h at The antibody, was in 0.5% fetal calf serum and incubated with the for 1 h at The was initiated with and the was in and exposed to Corp.). To the effects of farnesyl transferase inhibitors SCH 44342 and SCH 56582 J. L. G. J. J. J. James L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) on the prenylation of endogenous Ras proteins in a human cell line, the mevalonate transporter (17Kim C.M. Goldstein J.L. Brown M.S. J. Biol. Chem. 1992; 267: 23113-23121Abstract Full Text PDF PubMed Google Scholar) into DLD-1 cells. cells are from a colon carcinoma and normal K-Ras and mutant K-Ras of the mutant of to in soft and in nude mice S. M. N. Science. 1993; 260: PubMed Scopus Google of the mevalonate transporter in the DLD-1 cell line DLDpMev the cells to exogenous 3Hmevalonate in the of farnesyl and geranylgeranyl labeled in DLDpMev cells following in 100 μCi/ml 20 and of farnesyl transferase were by polyacrylamide gel followed by Treatment with of SCH 56582 mevalonate into several proteins in the cell lysate between and These proteins are to be farnesylated Maltese W.A. Biochem. 1991; PubMed Scopus Google Scholar). into proteins that a between and was not These proteins of a number of of the protein which are These are with the in of this compound for farnesyl protein transferase over geranylgeranyl transferase-1. Ras proteins were immunoprecipitated from the cell lysate with the pan-Ras of the mevalonate into immunoprecipitated Ras proteins was not by of SCH 56582 analysis of DLDpMev approximately using antibodies specific for N-Ras and K-Ras the H-Ras was to the N- and K-Ras not Y13–259 Ras the proteins immunoprecipitated in 1 are a of and We the of the prenyl group attached to the immunoprecipitated Ras proteins using methyl iodide and HPLC analysis (18Casey P.J. Solski P.A. Der C.J. Buss J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8323-8327Crossref PubMed Scopus (782) Google Scholar). prenyl groups from DLDpMev cells 2 to a in the from DLDpMev cells is to of the methyl iodide (18Casey P.J. Solski P.A. Der C.J. Buss J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8323-8327Crossref PubMed Scopus (782) Google Following with 20 the majority of prenyl groups with the 2 A for SCH 56582 is shown in 2 Ras proteins in DLDpMev cells are farnesylated in the absence of FTI but become geranylgeranylated in the presence of of SCH To determine with FTI have on of each of the Ras co-transfected COS cells with the mevalonate transporter pMEV (17Kim C.M. Goldstein J.L. Brown M.S. J. Biol. Chem. 1992; 267: 23113-23121Abstract Full Text PDF PubMed Google Scholar) and Ras N-Ras K-Ras4A K-Ras4B or the geranylgeranyl transferase-1 substrate H-Ras proteins were labeled by with 10 μmmevastatin and 100 μCi/ml and Ras proteins were immunoprecipitated with the pan-Ras Y13–259 K-Ras4A, N-Ras, and to 3Hmevalonate in the presence of 7.5 μm SCH H-Ras on the other is protein is expressed at in cells and cells treated with SCH 56582 and mevastatin that the of labeled H-Ras in 3 is to inhibition of prenylation and not to of H-Ras in the cell of COS cells were to in the cell of DLDpMev cells not of Ras proteins expressed in COS cells. COS-7 cell were following with Ras expression vectors into a (100,000 × g and a soluble protein × In H-Ras, K-Ras4A, N-Ras, and H-Ras-CVLL of proteins are and associate with the fraction. In from cells treated with mevastatin μm), of farnesyl and geranylgeranyl pyrophosphate is and of the Ras proteins as soluble Treatment with SCH 56582 μm) H-Ras from the to the soluble K-Ras4A, N-Ras, and H-Ras-CVLL associated with the fraction. of Ras proteins with with proteins the soluble analysis was performed on Ras from COS cells transfected with Ras expression vectors COS-7 cells were co-transfected with pMev and Ras expression and prenylated proteins were labeled and as described analysis of immunoprecipitated Ras proteins was done as described for In the absence of N-Ras, K-Ras4A, and H-Ras farnesyl This is to our in DLD-1 cells, Ras proteins were farnesylated in the absence of In the absence of K-Ras4B was found to a (approximately of the of geranylgeranyl with N-, H-, and This result was since have not of geranylgeranyl groups into K-Ras proteins in cells P.J. Solski P.A. Der C.J. Buss J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8323-8327Crossref PubMed Scopus (782) Google Scholar) and may be a of the high of expression of K-Ras4B in COS cells. In COS cells treated with 10 μm SCH 56582, of both N-Ras and K-Ras proteins H-Ras prenylation is by the to a of from the immunoprecipitated H-Ras These our observation of alternative prenylation in DLD-1 cells to cell COS-7 cells, and analysis of each of Ras to geranylgeranyl into K-Ras expressed in COS cells is in to the of James et G. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4454-4458Crossref PubMed Scopus (139) Google Scholar). farnesyl into H/K-Ras4BV12 expressed in Rat-1 cells in the presence or absence of a FTI. The in our may be to the cell or Ras the may be to concentration of mevalonate in our to of the mevalonate et al. E. L.M. Buss J.E. Biochem. 1993; PubMed Scopus Google Scholar) reported that protein prenylation in cells can as a function of the mevalonate The of SCH 56582 on and of the Ras proteins was by COS cells transfected with Ras expression vectors in the presence of 7.5 μm SCH 56582, 20 μmmevastatin a control for inhibition of or were into a (100,000 × g in and a soluble × of Ras can be by the of Ras proteins in polyacrylamide gels L. C.J. J.F. J. 1989; PubMed Scopus Google Scholar) and by the of Ras protein from the membrane fraction. In the absence of SCH 56582 or of each of the Ras proteins associated with the fraction. Treatment with mevastatin of forms of Ras from the and the of forms in the soluble fraction. In cells treated with SCH 56582, H-Ras is from the and in the soluble fraction. isoforms of in contrast, are as by and associated with the fraction. The with N-Ras in the presence of the farnesyl transferase are of the N-Ras protein expressed in COS cells is in the presence of SCH 56582 and associated with the fraction. of the N-Ras protein, as in the soluble have this inhibition of N-Ras prenylation in several both with and with These using analysis the using mevalonate N-Ras, K-Ras4A, and K-Ras4B are prenylated in the presence of FTI. that alternatively prenylated of N- and K-Ras associated with the membrane in treated cells. The in prenylation that in N- and K-Ras proteins in the presence of FTI is a of the of farnesyl protein transferase, with geranylgeranyl for J. Biol. in in the absence of N- and K-Ras proteins are farnesylated by farnesyl protein transferase. When farnesyl protein transferase is N- and K-Ras become as for geranylgeranyl transferase-1. H-Ras, on the other is not a substrate for geranylgeranyl transferase-1 and so The of H-Ras to inhibition that cells on H-Ras may be particularly to FTI In with have found that cells with mutant H-Ras are approximately sensitive to FTI in the soft agar cells with mutant K-Ras not al. (16James G. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4454-4458Crossref PubMed Scopus (139) Google Scholar) have Ras proteins a in as of transforming from mutant for and cells in which normal H-Ras a significant in oncogenic to be relatively sensitive to FTI The of H-Ras in the of cells treated with FTI may a in the mechanism of action of by normal signaling M. S. Y. 1996; PubMed Scopus Google Scholar). The alternative of N- and K-Ras proteins in the presence of FTI that they may to with proteins under In of have found in that, H-Ras of can be by farnesyl transferase inhibitors, and N-Ras of is in anchorage-independent growth of K-Ras and of human cells containing and N-Ras can be by FTI (9Kohl N.E. Mosser S.D. deSolms S.J. Giuliani E.A. Pompliano D.L. Graham S.L. Smith R.L. Scolnick E.M. Oliff A. Gibbs J.B. Science. 1993; 260: 1934-1937Crossref PubMed Scopus (620) Google Scholar). The mechanism by which FTIs anchorage-independent growth in cells is The of geranylgeranyl farnesyl into N- and K-Ras proteins may in Ras by or that to to in an anchorage-independent are stimulated by of Ras for membrane and M.H. Science. 1996; PubMed Scopus Google and is that alternative prenylation the mechanism of action of FTIs may inhibiting the of a protein other The mechanism of action of FTIs is The result of with FTI may the effects of inhibiting the prenylation of as H-Ras and the proteins in the DLDpMev lysate and the of prenylation of as N- and proteins that are farnesyl protein transferase may become geranylgeranylated farnesyl protein transferase is