Membrane Interactions of a Constitutively Active GFP-Ki-Ras 4B and Their Role in Signaling
1999; Elsevier BV; Volume: 274; Issue: 3 Linguagem: Inglês
10.1074/jbc.274.3.1606
ISSN1083-351X
AutoresHagit Niv, Orit Gutman, Yoav I. Henis, Yoel Kloog,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoMembrane anchorage of Ras proteins in the inner leaflet of the plasma membrane is an important factor in their signaling and oncogenic potential. Despite these important roles, the precise mode of Ras-membrane interactions is not yet understood. It is especially important to characterize these interactions at the surface of intact cells. To investigate Ras-membrane interactions in live cells, we employed studies on the lateral mobility of a constitutively active Ras isoform to characterize its membrane dynamics, and examined the effects of the Ras-displacing antagonistS-trans,trans-farnesylthiosalicylic acid (FTS) (Haklai, R., Gana-Weisz, M., Elad, G., Paz, A., Marciano, D., Egozi, Y., Ben-Baruch, G., and Kloog, Y. (1998) Biochemistry 37, 1306–1314) on these parameters. A green fluorescent protein (GFP) was fused to the N terminus of constitutively active Ki-Ras 4B(12V) to generate GFP-Ki-Ras(12V). When stably expressed in Rat-1 cells, this protein was preferentially localized to the plasma membrane and displayed transforming activity. The lateral mobility studies demonstrated that GFP-Ki-Ras(12V) undergoes fast lateral diffusion at the plasma membrane, rather than exchange between membrane-bound and unbound states. Treatment of the cells with FTS had a biphasic effect on GFP-Ki-Ras(12V) lateral mobility. At the initial phase, the lateral diffusion rate of GFP-Ki-Ras(12V) was elevated, suggesting that it is released from some constraints on its lateral mobility. This was followed by dislodgment of the protein into the cytoplasm, and a reduction in the diffusion rate of the fraction of GFP-Ki-Ras(12V) that remained associated with the plasma membrane. Control experiments with other S-prenyl analogs showed that these effects are specific for FTS. These results have implications for the interactions of Ki-Ras with specific membrane anchorage domains or sites. Membrane anchorage of Ras proteins in the inner leaflet of the plasma membrane is an important factor in their signaling and oncogenic potential. Despite these important roles, the precise mode of Ras-membrane interactions is not yet understood. It is especially important to characterize these interactions at the surface of intact cells. To investigate Ras-membrane interactions in live cells, we employed studies on the lateral mobility of a constitutively active Ras isoform to characterize its membrane dynamics, and examined the effects of the Ras-displacing antagonistS-trans,trans-farnesylthiosalicylic acid (FTS) (Haklai, R., Gana-Weisz, M., Elad, G., Paz, A., Marciano, D., Egozi, Y., Ben-Baruch, G., and Kloog, Y. (1998) Biochemistry 37, 1306–1314) on these parameters. A green fluorescent protein (GFP) was fused to the N terminus of constitutively active Ki-Ras 4B(12V) to generate GFP-Ki-Ras(12V). When stably expressed in Rat-1 cells, this protein was preferentially localized to the plasma membrane and displayed transforming activity. The lateral mobility studies demonstrated that GFP-Ki-Ras(12V) undergoes fast lateral diffusion at the plasma membrane, rather than exchange between membrane-bound and unbound states. Treatment of the cells with FTS had a biphasic effect on GFP-Ki-Ras(12V) lateral mobility. At the initial phase, the lateral diffusion rate of GFP-Ki-Ras(12V) was elevated, suggesting that it is released from some constraints on its lateral mobility. This was followed by dislodgment of the protein into the cytoplasm, and a reduction in the diffusion rate of the fraction of GFP-Ki-Ras(12V) that remained associated with the plasma membrane. Control experiments with other S-prenyl analogs showed that these effects are specific for FTS. These results have implications for the interactions of Ki-Ras with specific membrane anchorage domains or sites. The small G-proteins of the Ras family are essential components of signaling cascades that regulate important cell functions such as growth and differentiation (1Lange-Carter C.A. Johnson G.L. Science. 1994; 265: 1458-1461Crossref PubMed Scopus (296) Google Scholar, 2Cox A.D. Der C.J. Curr. Opin. Cell Biol. 1992; 4: 1008-1016Crossref PubMed Scopus (201) Google Scholar, 3Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1761) Google Scholar, 4Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1124) Google Scholar). Wild-type Ras isoforms alternate between inactive (Ras-GDP) and active (Ras-GTP) states (5Mittal R. Ahmadian M.R. Goody R.S. Wittinghofer A. Science. 1996; 273: 115-117Crossref PubMed Scopus (193) Google Scholar, 6Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1202) Google Scholar). Mutations at positions 12, 13, or 61 result in constitutively active Ras isoforms; these mutants bind GTP, have transforming activity, and contribute to uncontrolled cell growth (7Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3780) Google Scholar, 8Bos J.L. Eur. J. Cancer. 1995; 31: 1051-1054Abstract Full Text PDF Scopus (63) Google Scholar). The function of Ras proteins as signal transduction regulators and their oncogenic potential require association with the inner leaflet of the plasma membrane (2Cox A.D. Der C.J. Curr. Opin. Cell Biol. 1992; 4: 1008-1016Crossref PubMed Scopus (201) Google Scholar, 3Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1761) Google Scholar, 9Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (474) Google Scholar). Membrane anchorage of Ras proteins is promoted by their C-terminal S-farnesylcysteine and by either a stretch of lysines (Ki-Ras 4B) or by S-palmitoyl moieties (Ha- and N-Ras, or Ki-Ras 4A) (10Hancock J.F. Magee A.I. Childs J.E. Marshall C.J. Cell. 1989; 57: 1167-1177Abstract Full Text PDF PubMed Scopus (1460) Google Scholar, 11Casey P.J. Solski P.A. Der C.J. Buss J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8323-8327Crossref PubMed Scopus (780) Google Scholar, 12Kato K. Cox A.D. Hisaka M.M. Graham S.M. Buss J.E. Der C.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6403-6407Crossref PubMed Scopus (555) Google Scholar, 13Cox A.D. Hisaka M.M. Buss J.E. Der C.J. Mol. Cell. Biol. 1992; 12: 2606-2615Crossref PubMed Scopus (163) Google Scholar). The anchoring moieties of Ras proteins also appear to target them to the plasma membrane (2Cox A.D. Der C.J. Curr. Opin. Cell Biol. 1992; 4: 1008-1016Crossref PubMed Scopus (201) Google Scholar), possibly to specific membrane domains (14Song S.K. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar, 15Mineo C. James G.L. Smart E.J. Anderson R.G. J. Biol. Chem. 1996; 271: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). Although the essential role of membrane tethering in Ras signaling and transforming activity is well established, the precise mode of Ras-membrane interactions is not yet understood. It is not clear whether Ras proteins are stably associated with the plasma membrane or undergo rapid exchange between membrane-bound and unbound states (16Yokoe H. Meyer T. Nat. Biotech. 1996; 14: 1252-1256Crossref PubMed Scopus (165) Google Scholar), whether they form tight complexes with putative membrane receptors (17Siddiqui A.A. Garland J.R. Dalton M.B. Sinensky M. J. Biol. Chem. 1998; 273: 3712-3717Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and whether the Ras anchoring moieties (e.g.farnesylcysteine) interact randomly with the membrane lipid milieu or associate preferentially with distinctive domains or sites. A possible role for specific membrane domains is implied by the evidence that Ha-Ras is enriched in low buoyant density fractions typical of caveolae or analogous glycosphingolipid/cholesterol-enriched domains (14Song S.K. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar,15Mineo C. James G.L. Smart E.J. Anderson R.G. J. Biol. Chem. 1996; 271: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). We have recently developed compounds resembling the farnesylcysteine of Ras proteins (18Marciano D. Ben-Baruch G. Marom M. Egozi Y. Haklai R. Kloog Y. J. Med. Chem. 1995; 38: 1267-1272Crossref PubMed Scopus (110) Google Scholar, 19Marom M. Haklai R. Ben-Baruch G. Marciano D. Egozi Y. Kloog Y. J. Biol. Chem. 1995; 270: 22263-22270Crossref PubMed Scopus (170) Google Scholar, 20Aharonson Z. Gana-Weisz M. Varsano T. Haklai R. Marciano D. Kloog Y. Biochim. Biophys. Acta. 1998; 1406: 40-50Crossref PubMed Scopus (52) Google Scholar). One of these compounds,S-trans,trans-farnesylthiosalicylic acid (FTS), 1The abbreviations used are: FTS, S-trans,trans-farnesylthiosalicylic acid; AFC, N-acetyl-S-trans,trans-farnesyl-l-cysteine; BSA, bovine serum albumin; D , lateral diffusion coefficient; DiIC16, 1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocynanine perchlorate; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; FPR, fluorescence photobleaching recovery; GFP, green fluorescent protein; GFP-Ki-Ras(12V), GFP-tagged constitutively active Ki-Ras 4B(V12); GPI, glycosylphosphatidylinositol; GTS, S-geranylthiosalicylic acid; HBSS, Hanks' balanced salt solution; PBS, phosphate-buffered saline; R18, octadecyl rhodamine B chloride; RF , mobile fraction. inhibited the growth of cells transformed by Ha-Ras; the inhibition is not further downstream, since growth of cells transformed by constitutively active v-Raf was not affected (18Marciano D. Ben-Baruch G. Marom M. Egozi Y. Haklai R. Kloog Y. J. Med. Chem. 1995; 38: 1267-1272Crossref PubMed Scopus (110) Google Scholar, 19Marom M. Haklai R. Ben-Baruch G. Marciano D. Egozi Y. Kloog Y. J. Biol. Chem. 1995; 270: 22263-22270Crossref PubMed Scopus (170) Google Scholar). FTS specifically dislodged farnesylated Ha-Ras from membranes of Rat-1 cells, but not non-farnesylated N-myristoylated Ras or prenylated Gβγ of heterotrimeric G-proteins (21Haklai R. Gana-Weisz M. Elad G. Paz A. Marciano D. Egozi Y. Ben-Baruch G. Kloog Y. Biochemistry. 1998; 37: 1306-1314Crossref PubMed Scopus (189) Google Scholar), suggesting that it disrupts the interactions of Ras with specific anchorage domains. Since FTS appears to affect directly membrane-bound Ras, it can serve as a tool to investigate Ras-membrane interactions, and has a potential therapeutic value. Earlier studies on Ras-membrane interactions either employed cell-free systems or involved cell or tissue fixation. Obviously, it is important to characterize these interactions at the surface of intact cells. To investigate Ras-membrane interactions in live cells, we applied in the current work fluorescence photobleaching recovery (FPR) studies on the lateral mobility of green fluorescent protein (GFP)-tagged constitutively active Ki-Ras 4B(12V) (GFP-Ki-Ras(12V)) protein expressed in Rat-1 cells. In combination with experiments on the effects of the Ras antagonist FTS on Ras membrane anchorage, our results indicate that GFP-Ki-Ras(12V) undergoes fast lateral diffusion at the plasma membrane, rather than exchange between membrane-bound and unbound states. FTS was capable of releasing GFP-Ki-Ras(12V) from some constraints on its mobility during the early phase of FTS treatment, prior to the dislodgment of Ras from the plasma membrane. These results have implications for the nature of the interactions of Ki-Ras with specific membrane anchorage domains or sites. N-Acetyl-S-trans,trans-farnesyl-l-cysteine (AFC), S-geranylthiosalicylic acid (GTS) and FTS were prepared and purified as detailed elsewhere (18Marciano D. Ben-Baruch G. Marom M. Egozi Y. Haklai R. Kloog Y. J. Med. Chem. 1995; 38: 1267-1272Crossref PubMed Scopus (110) Google Scholar, 22Marciano D. Aharonson Z. Varsano T. Haklai R. Kloog Y. Bioorg. Med. Chem. Lett. 1997; 7: 1709-1714Crossref Scopus (13) Google Scholar). 1,1′-Dihexadecyl-3,3,3′,3′-tetramethylindocarbocynanine perchlorate (DiIC16) and octadecyl rhodamine B chloride (R18) were obtained from Molecular Probes (Eugene, OR). Bovine serum albumin (BSA), fatty acid-free BSA, Hanks' balanced salt solution (HBSS), cytochalasin D, peroxidase-conjugated goat anti-mouse or anti-rabbit IgG, and protein A-Sepharose 4B were from Sigma. Mouse monoclonal pan-Ras antibody-3 (anti-Ras) was purchased from Calbiochem, and anti-GFP rabbit polyclonal IgG was fromCLONTECH. The entire coding region of human Ki-Ras 4B(12V) cDNA cloned in pBluescript II SK via the PstI and BamHI sites of the multiple cloning region was a gift from P. Gierschik (University of Ulm, Ulm, Germany). The insertion was performed by the use of polymerase chain reaction to generate flanking sequences on the 5′ (CTGGAGCAT, containing aPstI site) and on the 3′ (GGATCC, a BamHI site) ends of the Ki-Ras(12V) coding region. It was then excised by the same restriction enzymes, and inserted intoPstI/BamHI-digested pEGFP-C3 (CLONTECH), resulting in a chimeric construct of enhanced GFP (a red-shifted enhanced GFP variant) fused in frame to the 5′ end of Ki-Ras(12V). The coding regions in the final construct were verified by DNA sequencing. Cells were routinely grown at 37 °C, 5% CO2, and 100% humidity, in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS; Biological Industries, Beth Haemek, Israel) and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). To generate stably expressing cell lines, Rat-1 cells (1 × 106 in a 35-mm dish) were transfected with 1 μg of DNA from the pEGFP-C3 vector (for expression of free, unfused GFP) or the same vector containing the GFP-Ki-Ras(12V) cDNA. The transfection mixture contained 10 μl of LipofectAMINE (Life Technologies, Inc.) in 1 ml of Opti-MEM (Life Technologies, Inc.). After 5 h of incubation at 37 °C, 1 ml of DMEM/FCS was added. After 48 h, the cells were transferred to a 10-cm dish, and incubated with selection medium (DMEM/FCS supplemented with 800 μg/ml G418; Life Technologies, Inc.). Single G418-resistant clones were isolated. The clones transfected with the vector containing the GFP-Ki-Ras(12V) cDNA were tested for expression of GFP-Ki-Ras(12V) (see "Results"), and two representative clones were selected for further analysis. For experiments on guanine nucleotide binding (see below), COS-7 cells were transiently transfected by the DEAE-dextran method (23Seed B. Aruffo A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3365-3369Crossref PubMed Scopus (789) Google Scholar) using 4 μg of the GFP-Ki-Ras(12V) construct per 1 × 106cells in a 10-cm dish. We have basically employed a protocol described previously (24Gibbs J.B. Marshall M.S. Scolnick E.M. Dixon R.A. Vogel U.S. J. Biol. Chem. 1990; 265: 20437-20442Abstract Full Text PDF PubMed Google Scholar, 25Satoh T. Endo M. Nakafuku M. Nakamura S. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5993-5997Crossref PubMed Scopus (200) Google Scholar). COS-7 cells were transfected as described above. Eighteen hours after transfection, the medium was replaced by serum-free DMEM, and incubation was continued for 18 h. After washing with DMEM devoid of serum and phosphate, the cells were incubated 4 h at 37 °C with 4 ml of the same medium supplemented with 0.5 mCi of carrier-free [32P]orthophosphate (Amersham). The cells were washed three times with ice-cold phosphate-buffered saline (PBS), and lysed (15 min, 4 °C) with 0.5 ml of lysis buffer (50 mmTris-HCl, pH 7.6, 20 mm MgCl2, 150 mm NaCl, 0.5% Nonidet P-40, 5 units/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin, 1 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride, 1 mmdithiothreitol). After removal of insoluble material (14,000 rpm, 10-min spin in Eppendorf centrifuge), free nucleotides were removed by 1.4% charcoal (24Gibbs J.B. Marshall M.S. Scolnick E.M. Dixon R.A. Vogel U.S. J. Biol. Chem. 1990; 265: 20437-20442Abstract Full Text PDF PubMed Google Scholar). From each sample, volumes containing equal amounts of radioactivity (5 × 107 cpm) were taken for immunoprecipitation, performed after completing the volume to 500 μl of lysis buffer, 1% BSA, 2 μg/ml anti-GFP. Following incubation for 2 h at 4 °C, 40 μl of protein A-Sepharose (120 μg of protein A) were added, and incubated for 1 h at 4 °C. The beads were washed twice in lysis buffer and once in PBS. The pellets were suspended in 20 μl of 20 mm Tris-HCl, pH 7.6, containing 20 mm EDTA, 2% SDS, 0.5 mm GDP, and 0.5 mm GTP. The suspension was heated (65 °C, 5 min) and centrifuged, and samples (11 μl) were spotted onto a polyethyleneimine cellulose TLC plate (Sigma), which was developed in 0.75 m KH2PO4, pH 3.4. The TLC plates were analyzed by a phosphorimager (FujiX Bas1000), and the spots corresponding to guanine nucleotides were quantified using TINA 2.0 software by Ray Test (Staubenhardt, Germany). Stock solutions of FTS and its analogs were freshly prepared in dimethyl sulfoxide (Me2SO) and diluted to working concentrations (10–50 μm) in DMEM supplemented with 10% FCS; the final Me2SO concentration was always 0.1%. Rat-1 cells expressing GFP-Ki-Ras(12V) or GFP were plated at densities specified for each experiment; 24 h later, the FTS-containing medium (or similar medium containing only 0.1% Me2SO in control experiments) was added for various time periods as indicated in the text. To estimate the effect of FTS on cell growth, the cells were plated in 24-well plates at 5000 cells/well, treated with the drug at various concentrations, and counted on day 4 of the treatment. Treatment with cytochalasin D (10 μg/ml, 15 min, 37 °C) was performed by adding it to the FTS-containing medium (or to the control medium) for the last 15 min of the incubation. The drug was kept in for the remainder of the experiment. Western blotting and ECL were performed as described by us previously (21Haklai R. Gana-Weisz M. Elad G. Paz A. Marciano D. Egozi Y. Ben-Baruch G. Kloog Y. Biochemistry. 1998; 37: 1306-1314Crossref PubMed Scopus (189) Google Scholar). Rat-1 cells expressing GFP-Ki-Ras(12V) or GFP were plated at a density of 1 × 106/10-cm dish. After FTS treatment as described above, they were homogenized, and the cytosolic (S100) and total membrane (P100) fractions were obtained by centrifugation (100,000 × g, 30 min, 4 °C) (21Haklai R. Gana-Weisz M. Elad G. Paz A. Marciano D. Egozi Y. Ben-Baruch G. Kloog Y. Biochemistry. 1998; 37: 1306-1314Crossref PubMed Scopus (189) Google Scholar). Samples of the total homogenate and of the S100 and P100fractions were calibrated for similar protein content, and subjected to SDS-polyacrylamide (12.5%) gel electrophoresis followed by electrophoretic blotting onto nitrocellulose filters. The filters were blocked and incubated with antibodies as detailed previously (21Haklai R. Gana-Weisz M. Elad G. Paz A. Marciano D. Egozi Y. Ben-Baruch G. Kloog Y. Biochemistry. 1998; 37: 1306-1314Crossref PubMed Scopus (189) Google Scholar), using either mouse anti-Ras (1:2000) or rabbit anti-GFP (1:1000) followed by peroxidase-goat anti-mouse (1:7500) or anti-rabbit (1:5000) IgG, respectively. Bands were visualized by ECL and quantified by densitometry on a BioImaging System 202D (Dynco-Renium, Jerusalem, Israel), using Tina 2.0 software (Ray Test). The lipophilic indocarbocyanine lipid analog DiIC16, which distributes equally into both the external and internal leaflets of the plasma membrane in cells (26Schootemeijer A. Van Beekhuizen A.E. Tertoolen L.G. de Laat S.W. Akkerman J.W. Eur. J. Biochem. 1994; 224: 423-430Crossref PubMed Scopus (18) Google Scholar), was incorporated into the plasma membrane of Rat-1 cells expressing GFP-Ki-Ras(12V) following the procedure of Edidin and Stroynowski (27Edidin M. Stroynowski I. J. Cell Biol. 1991; 112: 1143-1150Crossref PubMed Scopus (103) Google Scholar) with minor modifications. The cells were plated on glass coverslips placed in 35-mm dishes at 10,000 cells/dish. A stock solution of DiIC16 (100 μg/ml) was prepared in ethanol, and diluted prior to the experiment to 0.3 μg/ml in HBSS containing 20 mm HEPES, pH 7.4 (HBSS/HEPES). The cells were incubated with this labeling solution 10 min at 22 °C, washed three times with HBSS/HEPES supplemented with 0.1% fatty acid-free BSA to remove free dye, and immersed in HBSS/HEPES containing BSA (not fatty acid-free; HBSS/HEPES/BSA) and 0.1% Me2SO (same conditions as for FTS treatment) for FPR studies. In experiments where the cells were treated with FTS for 30 min or 24 h, they were washed with HBSS/HEPES prior to labeling, and FTS was added back at the end of the labeling procedure; other than that, the DiIC16 labeling was identical to that employed for untreated cells. Incorporation of the membrane lipid marker R18 into the cellular plasma membrane was performed following procedures described previously (28Hoekstra D. de Boer T. Klappe K. Wilschut J. Biochemistry. 1984; 23: 5675-5681Crossref PubMed Scopus (535) Google Scholar,29Morris S.J. Sarkar D.P. White J.M. Blumenthal R. J. Biol. Chem. 1989; 264: 3972-3978Abstract Full Text PDF PubMed Google Scholar). The procedure was similar to that described above for DiIC16, except that the labeling solution contained 1.8 μm R18, and the labeling was for 5 min at 4 °C. After the labeling, the cells were fixed with 4% paraformaldehyde in PBS, mounted in 0.1% gelatin/PBS, and visualized by confocal fluorescence microscopy. Lateral diffusion coefficients (D) and mobile fractions (RF) were measured by FPR (30Axelrod D. Koppel D.E. Schlessinger J. Elson E.L. Webb W.W. Biophys. J. 1976; 16: 1055-1069Abstract Full Text PDF PubMed Scopus (2033) Google Scholar, 31Koppel D.E. Axelrod D. Schlessinger J. Elson E.L. Webb W.W. Biophys. J. 1976; 16: 1315-1329Abstract Full Text PDF PubMed Scopus (349) Google Scholar) using previously described instrumentation (32Henis Y.I. Gutman O. Biochim. Biophys. Acta. 1983; 762: 281-288Crossref PubMed Scopus (31) Google Scholar). Cells were plated on glass coverslips at 10,000 cells/coverslip; they were treated where indicated with FTS or other drugs and/or labeled with DiIC16 as described above. The coverslip was placed (cells facing downward) over a serological slide with a depression filled with HBSS/HEPES/BSA and containing 0.1% Me2SO with or without FTS. The monitoring laser beam (Coherent Innova 70 argon ion laser; 488 nm and 1 microwatt for GFP fluorescence or 529.5 nm and 1 microwatt for DiIC16) was focused through the microscope (Zeiss Universal) to a Gaussian radius of 0.61 ± 0.02 μm using 100× oil immersion objective. In some cases, the beam size was changed to 1.23 ± 0.04 μm by using 40× water immersion objective. The beam radius was determined as described previously (33Petersen N.O. McConnaughey W.B. J. Supramol. Struct. Cell. Biochem. 1981; 17: 213-221Crossref PubMed Scopus (15) Google Scholar, 34Henis Y.I. Yaron T. Lamed R. Rishpon J. Sahar E. Katchalski Katzir E. Biopolymers. 1988; 27: 123-138Crossref PubMed Scopus (22) Google Scholar). The use of a pinhole in the image plane in the photometer head in front of the photomultiplier makes the light collection confocal, enabling the collection of fluorescence from a narrow depth in the focal plane (31Koppel D.E. Axelrod D. Schlessinger J. Elson E.L. Webb W.W. Biophys. J. 1976; 16: 1315-1329Abstract Full Text PDF PubMed Scopus (349) Google Scholar), focusing either on the plasma membrane or in the cytoplasm. A brief pulse (5 milliwatts, 30–40 ms for the 100× objective, and 40–80 ms for the 40× objective) bleached 50–70% of the fluorescence in the illuminated region. The time course of fluorescence recovery was followed by the attenuated monitoring beam. D andRF were extracted from the fluorescence recovery curves by nonlinear regression analysis (35Petersen N.O. Felder S. Elson E.L. Weir D.M. Herzenberg L.A. Blackwell C.C. Herzenberg L.A. Handbook of Experimental Immunology. Blackwell Scientific Publications, Edinburgh1986: 24.21-24.23Google Scholar). Incomplete fluorescence recovery is interpreted to represent fluorescence-labeled molecules immobile on the FPR experimental time scale (D ≤ 5 × 10−12 cm2/s). All the FPR measurements were conducted at 22 °C. In order to investigate the interactions of Ki-Ras with the plasma membrane of intact cells, we have generated a GFP-tagged constitutively active Ki-Ras 4B (GFP-Ki-Ras(12V)) and stably expressed it in Rat-1 cells (see "Experimental Procedures"). The orientation of the construct was chosen to retain an intact Ki-Ras C-terminal domain, which is required for post-translational modification and membrane anchorage. The GFP-Ki-Ras(12V) was properly synthesized in the cells, as evidenced by Western immunoblotting using either anti-Ras or anti-GFP antibodies (Fig. 1 A). Using anti-Ras, both GFP-Ki-Ras(12V) (54 kDa) and endogenous Ras (21 kDa) were detected, and only the latter was observed in homogenates prepared from cells expressing GFP alone. Upon labeling with anti-GFP, only the 54-kDa band was detected in cells expressing GFP-Ki-Ras(12V), and a 34-kDa band was observed in GFP-expressing Rat-1 cells. No labeling was detected in untransfected Rat-1 cells. The apparent molecular weight of GFP-Ki-Ras(12V) fitted that expected for a GFP-Ras fusion protein, and no smaller fragments were detected. To validate the constitutively active nature of GFP-Ki-Ras(12V), we employed a guanine nucleotide binding assay in intact cells. In this assay, we employed anti-GFP to immunoprecipitate GFP-Ki-Ras(12V), in order to ensure that the binding measured is to the fusion protein and not to endogenous Ras. This experiment (Fig. 1 B), performed in transiently transfected COS-7 cells, demonstrated preferential binding of GTP over GDP to GFP-Ki-Ras(12V) (80% versus 20%, respectively), as expected for a constitutively active Ras isoform (7Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3780) Google Scholar, 8Bos J.L. Eur. J. Cancer. 1995; 31: 1051-1054Abstract Full Text PDF Scopus (63) Google Scholar). The expression of GFP-Ki-Ras(12V) in the Rat-1 clones was further validated by confocal immunofluorescence microscopy. The fluorescent Ras isoform localized preferentially to the plasma membrane, as opposed to GFP expressed in Rat-1 cells, which distributed mainly to the cytoplasm and the nucleus (Fig. 2,A and B; see also Fig.3). This indicates that the association of GFP-Ki-Ras(12V) with the plasma membrane is mediated by the Ras protein in this construct and not by the GFP. Importantly, GFP-Ki-Ras(12V) is biologically active, as evidenced by its transforming activity. Thus, the GFP-Ki-Ras(12V)-expressing Rat-1 cell lines exhibited anchorage-independent growth in soft agar (Fig. 2,C and D). Furthermore, these cells were able to develop tumors in nude mice at a rate similar to that of cells expressing a constitutively active Ras (5 out of 5 mice in both cases).Figure 3Confocal microscopy demonstrates a shift of GFP-Ki-Ras(12V) from the plasma membrane to the cytoplasm following FTS treatment. Rat-1 cells stably expressing GFP-Ki-Ras(12V) were plated on glass coverslips, treated with 50 μm FTS for 48 h, and then labeled with R18 as described under "Experimental Procedures." After fixation with paraformaldehyde, dual images (green fluorescence for GFP, red for R18, andyellow where the two dyes coincide) were collected on the LSM 410 confocal microscope fitted with fluorescein and rhodamine filters. The images were exported in TIFF format to Adobe Photoshop and printed. Bar, 10 μm. The insets in each panel depict a z-scan analysis of the same cell (bar, 20 μm). A and D, GFP (green) fluorescence; B and E, R18 (red) fluorescence; C, superposition of the images in panels A and B; F, superposition of the images inpanels D and E. A–C, no FTS treatment (48-h incubation in medium with 0.1% Me2SO). D–F, FTS-treated cells.View Large Image Figure ViewerDownload (PPT) We have recently demonstrated that FTS, a compound resembling farnesylcysteine, specifically dislodges farnesylated Ha-Ras(12V) from membranes of Rat-1 cells (21Haklai R. Gana-Weisz M. Elad G. Paz A. Marciano D. Egozi Y. Ben-Baruch G. Kloog Y. Biochemistry. 1998; 37: 1306-1314Crossref PubMed Scopus (189) Google Scholar). To examine the effect of FTS on GFP-Ki-Ras(12V), Rat-1 cells stably expressing the fusion protein were subjected to FTS treatment and to analysis by confocal fluorescence microscopy. The results of a typical experiment are depicted in Fig. 3. Prior to FTS treatment, GFP-Ki-Ras(12V) was localized mainly at the rim of the cells, exhibiting typical plasma membrane labeling and co-localization with R18, a fluorescent membrane marker (Fig. 3, panels A–C; see also the insetsfor optical scanning along the z axis). Following FTS treatment (50 μm, 24–48 h), a significant amount of GFP-Ki-Ras(12V) was dislodged from the membrane into the cytoplasm; the dislodgment was not complete, leaving some GFP-Ki-Ras(12V) at the plasma membrane (Fig. 3, panels D–F; see insetsfor z-scan). As expected, R18 remained at the plasma membrane. The dislodgment of GFP-Ki-Ras(12V) from the membrane was also followed biochemically, quantifying GFP-Ki-Ras(12V) in the membrane pellet and in the cytosolic fractions by immunoblotting with anti-GFP antibodies. Incubation of Rat-1 cells expressing GFP-Ki-Ras(12V) with 50 μm FTS for 24 h mediated a reduction in the relative amount of the protein associated with the membranes and a parallel increase in its cytosolic fraction, although a significant fraction remained in the membrane pellet (Fig. 4). This suggests that GFP-Ki-Ras(12V) is dislodged by FTS from the membrane and accumulates in the cytoplasm, in accord with the confocal microscopy results (Fig. 3). This differs from our former observations on the fate of Ha-Ras(12
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