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• W1985753931 abstract "We recently identified a prenyl peptide-binding protein in microsomal membranes from bovine brain (Thissen, J. A., and Casey, P. J. (1993)J. Biol. Chem. 268, 13780–13783). Through a variety of approaches, this binding protein has been identified as the cytoskeletal protein tubulin. Prenyl peptides bind to purified tubulin with a K d of 40 nm and also bind to tubulin polymerized into microtubules. Microtubule affinity chromatography of extracts from cells in which the prenyl protein pool was metabolically labeled revealed that prenyl proteins bound to the immobilized microtubules; one, a 24-kDa protein, was tentatively identified as a GTP-binding protein. Of several prenylated GTP-binding proteins tested, including Ki-Ras4B, Ha-Ras, RhoB, RhoA, and Rap1B, only Ki-Ras was found to bind significantly to microtubules, and this was in a prenylation-dependent fashion. A potential significance of the interaction of Ki-Ras4B with microtubules was indicated from analysis of the localization of newly synthesized Ki-Ras4B and Ha-Ras, each tagged with green fluorescence protein (GFP). Treatment of NIH-3T3 cells expressing GFP-Ki-Ras with Taxol (paclitaxel) resulted in accumulation of the expressed protein in intracellular locations, whereas in control cells the protein was correctly targeted to the plasma membrane. Importantly, such treatment with paclitaxel did not affect the cellular localization of expressed GFP-Ha-Ras. These results indicate that an intact microtubule network may be directly involved in Ki-Ras processing and/or targeting and provide direct evidence for a physiological distinction between Ki-Ras and Ha-Ras in cells. Additionally, the finding that paclitaxel treatment of cells disrupts Ki-Ras trafficking suggests an additional mechanism for the anti-proliferative effects of this drug. We recently identified a prenyl peptide-binding protein in microsomal membranes from bovine brain (Thissen, J. A., and Casey, P. J. (1993)J. Biol. Chem. 268, 13780–13783). Through a variety of approaches, this binding protein has been identified as the cytoskeletal protein tubulin. Prenyl peptides bind to purified tubulin with a K d of 40 nm and also bind to tubulin polymerized into microtubules. Microtubule affinity chromatography of extracts from cells in which the prenyl protein pool was metabolically labeled revealed that prenyl proteins bound to the immobilized microtubules; one, a 24-kDa protein, was tentatively identified as a GTP-binding protein. Of several prenylated GTP-binding proteins tested, including Ki-Ras4B, Ha-Ras, RhoB, RhoA, and Rap1B, only Ki-Ras was found to bind significantly to microtubules, and this was in a prenylation-dependent fashion. A potential significance of the interaction of Ki-Ras4B with microtubules was indicated from analysis of the localization of newly synthesized Ki-Ras4B and Ha-Ras, each tagged with green fluorescence protein (GFP). Treatment of NIH-3T3 cells expressing GFP-Ki-Ras with Taxol (paclitaxel) resulted in accumulation of the expressed protein in intracellular locations, whereas in control cells the protein was correctly targeted to the plasma membrane. Importantly, such treatment with paclitaxel did not affect the cellular localization of expressed GFP-Ha-Ras. These results indicate that an intact microtubule network may be directly involved in Ki-Ras processing and/or targeting and provide direct evidence for a physiological distinction between Ki-Ras and Ha-Ras in cells. Additionally, the finding that paclitaxel treatment of cells disrupts Ki-Ras trafficking suggests an additional mechanism for the anti-proliferative effects of this drug. Post-translational addition of isoprenoid lipids via a process termed prenylation is important in the maturation of many proteins that play critical roles in signal transduction and cell growth regulation. In many cases, a requirement for the attached isoprenoid for correct cellular localization and function has been demonstrated (for reviews, see Refs. 1Marshall C.J. Science. 1993; 259: 1865-1866Crossref PubMed Scopus (302) Google Scholar, 2Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1733) Google Scholar, 3Der C.J. Cox A.D. Cancer Cells. 1991; 3: 331-340PubMed Google Scholar). Most prenylation events involve addition of either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid to a conserved Cys residue in a C-terminal sequence termed a “CAAX motif.” The X residue of the CAAX motif generally specifies which isoprenoid is to be attached to the protein by one of two cytosolic protein prenyltransferases, designated protein farnesyltransferase and protein geranylgeranyltransferase type I (4Casey P.J. Thissen J.A. Moomaw J.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8631-8635Crossref PubMed Scopus (156) Google Scholar, 5Reiss Y. Goldstein J.L. Seabra M.C. Casey P.J. Brown M.S. Cell. 1990; 62: 81-88Abstract Full Text PDF PubMed Scopus (702) Google Scholar, 6Yokoyama K. Goodwin G.W. Ghomashchi F. Glomset J.A. Gelb M.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5302-5306Crossref PubMed Scopus (217) Google Scholar). The addition of the isoprenoid lipid is followed by the proteolytic removal of the three C-terminal amino acids (the AAX) and methylation of the now C-terminal prenylated Cys residue in a process generally thought to occur in the microsomal membrane compartment of cells (7Jang G.F. Yokoyama K. Gelb M.H. Biochemistry. 1993; 32: 9500-9507Crossref PubMed Scopus (37) Google Scholar, 8Ashby M.N. King D.S. Rine J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4613-4617Crossref PubMed Scopus (98) Google Scholar, 9Perez-Sala D. Tan E.W. Canada F.J. Rando R.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3043-3046Crossref PubMed Scopus (108) Google Scholar, 10Hancock J.F. Cadwallader K. Marshall C.J. EMBO J. 1991; 10: 641-646Crossref PubMed Scopus (249) Google Scholar). In addition to this sequence of protein modifications, many prenyl proteins contain either an upstream polybasic region or an attached palmitoyl lipid near the C terminus that influences membrane binding (11Kato 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, 12Hancock J.F. Paterson H. Marshall C.J. Cell. 1990; 63: 133-139Abstract Full Text PDF PubMed Scopus (844) Google Scholar). Several studies implicate prenylated proteins in regulation of cytoskeletal events. Two members of the Ras superfamily of G proteins, Rap1B and Rap1A, associate with the cytoskeleton during agonist-stimulated platelet activation (13Torti M. Ramaschi G. Sinigaglia F. Lapetina E.G. Balduini C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7553-7557Crossref PubMed Scopus (40) Google Scholar, 14Fischer T.H. Gatling M.N. McCormick F. Duffy C.M. White G.C. II J. Biol. Chem. 1994; 269: 17257-17261Abstract Full Text PDF PubMed Google Scholar). The related proteins Rac and Rho are thought to be important regulators of actin filament organization. Evidence to support this hypothesis comes primarily from studies demonstrating that Rac and Rho, respectively, are involved in regulation of growth factor-induced membrane ruffling and assembly of focal adhesion and actin stress fibers in Swiss 3T3 cells (15Ridley A.J. Paterson H.F. Johnston C.L. Diekman D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3071) Google Scholar, 16Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3824) Google Scholar). Rac1 has also been found to associate with tubulin in the GTP-bound form, but the physiological significance is unknown (17Best A. Ahmed S. Kozma R. Lim L. J. Biol. Chem. 1996; 271: 3756-3762Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The role of prenylation in these processes and interactions is unclear; however, association of RHO1, the Saccharomyces cerevisiae equivalent of the mammalian RhoA, with cortical actin patches requires that it be prenylated for this function (18Yamochi W. Tanaka K. Nonaka H. Maeda A. Musha T. Takai Y. J. Cell. Biol. 1994; 125: 1077-1093Crossref PubMed Scopus (209) Google Scholar). There is also precedence in the literature for involvement of prenylated proteins in microtubule-dependent processes. For example, morphological changes induced by oncogenic Ras transformation can be profoundly influenced by vinca alkaloids, which promote microtubule breakdown, and Taxol (paclitaxel), which promotes formation of short bundled microtubules (19Gloushankova N.A. Lyubimova A.V. Tint I.S. Feder H.H. Vasilev J.M. Gelfand I.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8597-8601Crossref PubMed Scopus (25) Google Scholar, 20Rowinsky E.K. Donehower R.C. Pharmacol. Ther. 1991; 52: 35-84Crossref PubMed Scopus (317) Google Scholar, 21Olah E. Csokay B. Prajda N. Kote-Jarai Z. Yeh Y.A. Weber G. Anticancer Res. 1996; 16: 2469-2478PubMed Google Scholar). In one of these studies, epithelial cells transformed with oncogenic N-Ras exhibited a distinct polarized cell morphology that was abolished by treatment of the cells with either colcemid or paclitaxel (19Gloushankova N.A. Lyubimova A.V. Tint I.S. Feder H.H. Vasilev J.M. Gelfand I.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8597-8601Crossref PubMed Scopus (25) Google Scholar). Additional evidence for an interaction between prenylated proteins and cellular processes involving microtubules comes from studies using hydroxymethylglutaryl-CoA reductase inhibitors. These inhibitors, such as lovastatin and compactin, ultimately inhibit the synthesis of prenylated proteins and produce distinct changes in cell morphology (22Endo A. J. Lipid Res. 1992; 33: 1569-1582Abstract Full Text PDF PubMed Google Scholar, 23Schmidt R.A. Glomset J.A. Wight T.N. Habenicht A.J. Ross R. J. Cell Biol. 1982; 95: 144-153Crossref PubMed Scopus (54) Google Scholar). In one study, the appearance of the morphological changes in epithelial cells induced by compactin was found to coincide with the retraction of the microtubules from the submembrane regions (24Bifulco M. Laezza C. Aloj S.M. Garbi C. J. Cell. Physiol. 1993; 155: 340-348Crossref PubMed Scopus (52) Google Scholar). Pretreatment of the cells with colchicine to depolymerize the microtubules before treatment with compactin prevented these changes, suggesting that intact microtubules were necessary for the compactin-induced morphological changes observed (24Bifulco M. Laezza C. Aloj S.M. Garbi C. J. Cell. Physiol. 1993; 155: 340-348Crossref PubMed Scopus (52) Google Scholar). It is now established that mature prenylated proteins are associated with many different membrane compartments in cells, including plasma and intracellular membranes, and the cytoskeleton (12Hancock J.F. Paterson H. Marshall C.J. Cell. 1990; 63: 133-139Abstract Full Text PDF PubMed Scopus (844) Google Scholar, 25Casey P.J. Science. 1995; 268: 221-225Crossref PubMed Scopus (729) Google Scholar, 26James G.L. Goldstein J.L. Pathak R.K. Anderson R.G.W. Brown M.S. J. Biol. Chem. 1994; 269: 14182-14190Abstract Full Text PDF PubMed Google Scholar, 27Ramaschi G. Balduini C. Torti M. Sinigaglia F. Biochim. Biophys. Acta. 1994; 1199: 20-26Crossref PubMed Scopus (7) Google Scholar). However, little information is available on how newly prenylated proteins are directed from the cytosol to sites for subsequent processing and, ultimately, to their final destination, and it seems likely that additional factors must be involved in the correct targeting of these proteins. In attempts to identify factors involved in this process, we initiated a search for proteins that bind prenylated peptides encompassing the C termini of known prenylated proteins. This ligand binding approach resulted in the identification of a specific binding site in a microsomal membrane fraction that recognized both farnesyl- and geranylgeranyl-modified peptides (28Thissen J.A. Casey P.J. J. Biol. Chem. 1993; 268: 13780-13783Abstract Full Text PDF PubMed Google Scholar). The properties and specificity of this binding site were consistent with those expected for a molecule involved in targeting newly prenylated proteins to the subcellular compartment where their processing is completed. We have now extended those analysis to a molecular identification of the binding protein. Surprisingly, the major cellular protein identified that specifically binds a prenylated peptide was the microtubule protein tubulin, which comprises a significant part of the cell's cytoskeletal structure. A variety of experimental approaches indicate a physiological significance for the association of a specific prenylated protein, that being Ki-Ras4B with microtubules. The relationship between these findings and specific microtubule-dependent processes and prenyl protein trafficking is discussed. [1-3H]Farnesyldiphosphate ([3H]FPP 1The abbreviations used are: FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; MVA, mevalonolactone; BS3, bis(sulfosuccinimidyl)suberate; DTT, dithiothreitol; CHO, Chinese hamster ovary; Pipes, 1,4-piperazinediethanesulfonic acid; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; MAP kinase, mitogen-activated protein kinase. ; 22 Ci/mmol), [1-3H]geranylgeranyldiphosphate ([3H]GGPP; 15 Ci/mmol) and [α-32P]GTP (3000 Ci/mmol) were purchased from NEN Life Science Products. [3H]Mevalonolactone ([3H]MVA, 35 Ci/mmol) and unlabeled FPP and GGPP were obtained from American Radiolabeled Chemicals (St. Louis, MO). Peptides were synthesized on a Synergy peptide synthesizer (Applied Biosystems Inc.) and purified by high performance liquid chromatography before use. Activated CH-Sepharose 4B and glutathione-Sepharose 4B was obtained from Pharmacia Biotech Inc. Ni-NTA resin was from Qiagen (Chatsworth, CA). The BS3cross-linking reagent was from Pierce (Rockford, IL). Trifluoroacetic acid and iodomethane were from Aldrich. Affi-Gel 10 resin was from Bio-Rad. Protogel™ was from National Diagnostics (Atlanta, GA). Ham's F-12 medium was from Bio Whittaker (Walkersville, MD), and all other cell culture reagents were from Life Technologies, Inc. Lovastatin was a generous gift from Al Alberts (Merck, Rahway, NJ). Bacterial expression plasmids for Ha-Ras and Ki-Ras4B were gifts from Channing Der (University of North Carolina, Chapel Hill, NC (UNC-Chapel Hill)) and Ana Maria Garcia (Eisai Research Institute, Andover, MA), respectively, and the bacterial expression plasmid for Rap1B was from Guy James (University of Texas Southwestern Medical Center, Dallas, TX). RhoA was provided as a glutathione S-transferase fusion protein by Charles Minkoff (Duke University Medical Center, Durham, NC), and RhoB was provided by Peter Lebowitz (The Wistar Institute, Philadelphia, PA). NIH-3T3 cells stably transfected with Ha-Ras was provided by Adrienne Cox (UNC-Chapel Hill). Bovine brains were fractionated into cytosolic microsomal membrane- and plasma membrane-enriched fractions by differential centrifugation. Homogenates, prepared as described (28Thissen J.A. Casey P.J. J. Biol. Chem. 1993; 268: 13780-13783Abstract Full Text PDF PubMed Google Scholar), were centrifuged first at 16,900 × g for 60 min at 4 °C to collect plasma membranes and then at 100,000 ×g for 60 min at 4 °C to obtain cytosolic (supernatant) and microsomal membrane (pellet) fractions. Membrane fractions were resuspended at protein concentrations of 1–2 mg/ml in 20 mm Hepes, pH 7.0, 1 mm EDTA and 1 mm dithiothreitol (DTT) (Buffer A) containing a mixture of protease inhibitors (29Moomaw J.F. Zhang F.L. Casey P.J. Methods Enzymol. 1995; 250: 12-21Crossref PubMed Scopus (25) Google Scholar). Both the cytosolic and membrane fractions were flash-frozen in liquid nitrogen and stored at −80 °C until use (28Thissen J.A. Casey P.J. J. Biol. Chem. 1993; 268: 13780-13783Abstract Full Text PDF PubMed Google Scholar). The peptide corresponding essentially to the C terminus of a major G protein γ subunit (sequence: REKKFFCAIM) was enzymatically prenylated using [3H]FPP (15–22.5 Ci/mmol) and purified protein farnesyltransferase (28Thissen J.A. Casey P.J. J. Biol. Chem. 1993; 268: 13780-13783Abstract Full Text PDF PubMed Google Scholar). A typical reaction mixture contained 400 pmol of the peptide, 200 pmol of [3H]FPP, and 200 units of enzyme in 30 μl of 50 mm Tris-HCl, pH 8.0, 20 mm KCl, 5 mm MgCl2, 5 μm ZnCl2, and 2 mm DTT (4Casey P.J. Thissen J.A. Moomaw J.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8631-8635Crossref PubMed Scopus (156) Google Scholar). The reaction was initiated with enzyme and incubated 20 min at 37 °C, whereupon octylglucoside was added to a final concentration of 0.1% (to stabilize the prenylated peptide) and the reaction allowed to continue for an additional 60 min. The 3H-farnesylated peptide product was purified on a C18 reverse-phase HPLC column (Phenomenex), lyophilized, and stored at −20 °C until use. All GTP-binding proteins were produced in Escherichia coli. Ha-Ras and RhoB were purified as described (4Casey P.J. Thissen J.A. Moomaw J.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8631-8635Crossref PubMed Scopus (156) Google Scholar), and glutathioneS-transferase-RhoA was purified using glutathione-Sepharose 4B (Pharmacia Biotech Inc.) according to the manufacturers, and (His)6-Ki-Ras and and (His)6-Rap1B were purified using Ni-NTA resin (Qiagen) according to the manufacturer. Each GTP-binding protein was enzymatically prenylated by the appropriate isoprenoid as described previously (4Casey P.J. Thissen J.A. Moomaw J.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8631-8635Crossref PubMed Scopus (156) Google Scholar). Two different methods were used to chemically prenylate peptides used in this study. The first method provides a yield of ∼30–50% of the prenyl peptide product and was used in the initial part of this study, whereas the second method results in >90% product formation and was used in the latter part of this work. In the first method, peptides were chemically prenylated essentially as described (30Stephenson R.C. Clarke S. J. Biol. Chem. 1990; 265: 16248-16254Abstract Full Text PDF PubMed Google Scholar), as modified by Robert Deschenes (University of Iowa). 2R. Deschenes, personal communication. Briefly, 0.9 mg of peptide was dissolved in 1.26 ml of H2O:acetonitrile (1:5) on ice, followed by the addition of 210 μl of 0.5 mNaHCO3. The solution was placed under argon, and the prenylation reaction initiated by addition of farnesyl bromide in Me2SO (1.2 mol of farnesyl bromide/mol of peptide). The reaction was stirred at 4 °C for 3–4 h, and the product was then purified by reverse-phase HPLC, lyophilized, and stored at −20 °C. The second method involved dissolving the peptide and farnesyl bromide at a 1:1 molar ratio in methanol containing 4 m ammonia (31Brown M.J. Milano P.D. Lever D.C. Epstein W.W. Poulter C.D. J. Am. Chem. Soc. 1991; 113: 2176-3177Google Scholar). After a 3-h incubation at 4 °C, the reaction was dried under vacuum and the product purified by HPLC. Chemical cross-linking analysis was performed essentially as described (32Thissen J.A. Barrett M.G. Casey P.J. Methods Enzymol. 1995; 250: 158-168Crossref PubMed Scopus (4) Google Scholar). Briefly, bovine brain microsomal membranes or cytosol (each at 12 μg of protein) were incubated with 120–133 nm3H-labeled prenyl peptide in 150 μl of Buffer A plus 0.05% Triton X-100. After a 20-min incubation at 24 °C, BS3 cross-linking reagent was added to the indicated concentration, the incubation continued for 2 min, and the reaction quenched by the addition of 50 mmTris-Cl, pH 8.0. Protein was precipitated with trichloroacetic acid at 15% final concentration, and the precipitated protein containing the chemically cross-linked prenylated peptide-protein complex was collected by microcentrifugation and washed with ice-cold acetone. Precipitated proteins were then suspended in Laemmli sample buffer and subjected to SDS-PAGE (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar), and the resulting gels processed for fluorographic analysis as described (32Thissen J.A. Barrett M.G. Casey P.J. Methods Enzymol. 1995; 250: 158-168Crossref PubMed Scopus (4) Google Scholar). A 50-nmol amount of either the farnesylated peptide or the corresponding unprenylated peptide was dissolved 20 mm Hepes, pH 8.0, containing 50% dimethylformamide. To this was added 0.3 mg of activated CH-Sepharose 4B, which had been prepared as per the manufacturer's instructions. The mixture was incubated at room temperature for 1 h, followed by an overnight incubation with mixing at 4 °C. The resin was then poured into a 2-ml column and extensively washed using alternating pH buffers as described by the manufacturer. The column was stored in 50 mm sodium acetate, pH 4.0, containing 500 mmNaCl, 0.05% Triton X-100, and 0.025% NaN3 at 4 °C until use. Tubulin was purified from extracts of freshly isolated porcine brain essentially as described (34Simon J.R. Adam N.A. Salmon E.D. Micron. Microsc. Acta. 1991; 22: 405-412Crossref Scopus (10) Google Scholar). Briefly, a porcine brain (∼220 g) was homogenized at a ratio of 0.5 ml of buffer/g of brain tissue in 100 mmPipes, pH 6.9, containing 2 mm EGTA and 1 mmMgSO4 (PEM buffer), which also contained 1 mmATP and the protease inhibitors noted above. The homogenate was centrifuged at 100,000 × g for 1 h at 4 °C to remove membranes and organelles, and the supernatant diluted 1:1 with PEM buffer containing 60% glycerol and 0.2 mm GTP. After a 45-min incubation at 37 °C to polymerize tubulin, microtubules were collected by centrifugation at 100,000 × g for 45 min at 29 °C. The pellet containing the microtubules was then processed through a second depolymerization-polymerization by cycling between 4 °C and 37 °C. The two-cycle purified tubulin was then purified to >99% homogeneity using phosphocellulose chromatography as described (35Voter W.A. Erickson H.P. J. Biol. Chem. 1984; 259: 10430-10438Abstract Full Text PDF PubMed Google Scholar). Purified tubulin was stored in aliquots at −80 °C until use. Purified tubulin (50 ng) was incubated with 3H-farnesylated peptide (8–600 nm) in 10 mm Hepes, pH 7.0, containing 1 mm EGTA, 0.5 mm MgCl2, 50 mm NaCl, and 0.01% octylglucoside (Buffer B) in a volume of 50 μl. After a 20-min incubation at room temperature, samples were applied to a column containing 1 ml of Sephadex G-50 and washed with 0.5-ml aliquots of Buffer B. Tubulin-bound 3H-farnesylated peptide eluted in the void fraction and was detected by liquid scintillation spectroscopy. Specific binding was determined by the difference between 3H-farnesylated peptide bound to tubulin in the absence and presence of 2 μm unlabeled prenyl peptide. Microtubule affinity columns were constructed essentially as described (36Kellog D.R. Field C.M. Alberts B.M. J. Cell Biol. 1989; 109: 2977-2991Crossref PubMed Scopus (112) Google Scholar). Briefly, tubulin (2–3 mg/ml) in 80 mm Pipes, pH 6.8, 1 mm MgCl2, 1 mm Na3EGTA was assembled into microtubules by adding 1 mm GTP, followed by the stepwise addition of paclitaxel. Immediately before applying the sample to the activated resin, the pH of the solution containing the paclitaxel-stabilized microtubules was adjusted to 7.6 with 3 m KOH and a volume of the paclitaxel-stabilized microtubule solution equivalent to one-half of the resin volume added to the resin. The mixture was left undisturbed overnight at 4 °C to allow coupling, and then washed with several column volumes of 50 mm Hepes, pH 7.5, 1 mm EGTA, and 1 mm MgCl2 (Buffer C), containing 10 mm ethanolamine to block unreacted groups. The resin was then washed exhaustively with Buffer C containing 1 mm DTT and 500 mm KCl. Typical tubulin concentration obtained on the column was 6–9 μm. The column was stored at 4 °C in the assembly buffer containing 10% glycerol, 1 mm DTT, 5 μm paclitaxel, 0.02% sodium azide, and protease inhibitors. Stock cultures of the met18b-2 variant of Chinese hamster ovary (CHO) cells (37Faust J. Krieger M. J. Biol. Chem. 1987; 262: 1996-2004Abstract Full Text PDF PubMed Google Scholar) were grown in Ham's F-12 medium containing 100 units/ml penicillin, 100 units/ml streptomycin, and 2 mm glutamine and supplemented with 5% (v/v) fetal calf serum (supplemented Ham's F-12 medium) at 37 °C in 5% CO2. Cells were seeded at 3 × 105cells/100-mm dish and grown to about 70% confluence. In preparation for [3H]mevalonic acid labeling, cells were incubated in 5 ml of supplemented F-12 medium containing 30 μmlovastatin for 60–90 min at 37 °C. After this treatment, the medium was replaced by 5 ml of supplemented F-12 medium containing 250 μCi of [3H]mevalonolactone and 20 μm lovastatin and incubated overnight at 37 °C. Cell were harvested by scraping with a rubber policeman into Buffer C containing protease inhibitors and either used immediately or flash-frozen in liquid N2and stored at −80 °C until use. Stock cultures of NIH-3T3 cells were grown in Dulbecco's minimum essential medium with 10% fetal bovine serum (Life Sciences, Inc.), 1 mml-glutamine and 10 mg/ml gentamycin at 37 °C and 5% CO2. For use in microtubule affinity chromatography analysis, cells were lysed by the addition of Triton X-100 to 1% and NaCl to 250 mm, followed by homogenization on ice for 30 min and then centrifugation at 100,000 × g for 1 h at 4 °C. The supernatants were diluted 5-fold with Buffer C to reduce the detergent and NaCl, immediately applied to a microtubule affinity column equilibrated with Buffer C containing 50 mm NaCl and 0.05% Nonidet-40, and chromatographed as described in the figure legends. Ha-Ras, Ki-Ras, RhoA, RhoA, and Rap1B proteins were enzymatically prenylated with [3H]prenyldiphosphates as described above. Initially, 3H-farnesylated Ha-Ras and Ki-Ras were produced and chromatographed through a microtubule affinity column as described above for the [3H]MVA-labeled CHO cell extracts, except that 1 mg of cytochrome c was added to the Ras sample as a carrier protein. In later analysis, the3H-prenylated proteins were incubated with 100 μl (bed volume) of the microtubule affinity resin, prepared as described above, for 30 min at room temperature in Buffer C containing 50 mmNaCl and 0.05% Triton X-100. After the incubation, samples were centrifuged 4 min at 4 °C and washed twice with Buffer C containing 50 mm NaCl and 0.05% Triton X-100. The washed resin was then suspended in Laemmli sample buffer and analyzed by 14% SDS-PAGE/fluorography as described below. Most electrophoresis was performed essentially according to Laemmli (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar). Electrophoretic separation of the α and β subunits of tubulin was performed by SDS-PAGE using a 7.5% gel polymerized in the presence of 8m urea (38Eipper B.A. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 2283-2287Crossref PubMed Scopus (198) Google Scholar). Several different antibodies directed against tubulin were used in immunoblot analysis. These included an α tubulin-directed monoclonal antibody from ICN, a polyclonal antibody directed against residues 428–437 of the β subunit (a gift from M. Rasenick (University of Illinois, Chicago, IL), and two polyclonal antiserum directed against C-terminal sequences (both gifts from G. Gundersen, Columbia University, New York, NY; Ref. 39Gundersen G.G. Kalnoski M.H. Bulinski J.C. Cell. 1984; 38: 779-789Abstract Full Text PDF PubMed Scopus (380) Google Scholar). Antibody binding was detected using the alkaline phosphatase method (Promega). Immunoblot analysis of Ras proteins was performed using monoclonal antibody Y13-259 (Santa Cruz Biotechnology, Inc.). GTP binding was determined by an overlay assay on nitrocellulose essentially as described (40Bhullar R.P. Haslam R.J. Biochem. J. 1987; 245: 617-620Crossref PubMed Scopus (132) Google Scholar). Proteins were separated by SDS-PAGE and transferred to nitrocellulose. The nitrocellulose blot was first washed in 50 mm Tris-Cl, pH 7.5, containing 50 μm MgCl2 and 0.3% Tween 20 (Buffer D) for 5 min. The washed blot was then incubated in Buffer D containing 1 μCi/ml [α-32P]GTP and 10 μm ATP for 2–3 h. After the incubation, the blot was washed six times for 10 min each in Buffer D containing 5 mm MgCl2, dried, and exposed to Fuji RX film with an intensifying screen. Isoprenoids attached to proteins were determined by HPLC analysis of the methyl iodide cleavage product essentially as described (41Casey P.J. Solski P.A. Der C.J. Buss J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8323-8327Crossref PubMed Scopus (779) Google Scholar, 42Farnsworth C.C. Casey P.J. Howald W.N. Glomset J.A. Gelb M.H. Methods: Companion Methods Enzymol. 1990; 1: 231-240Crossref Scopus (27) Google Scholar). Briefly, samples containing3H-labeled prenyl proteins were precipitated with 15% trichloroacetic acid, and the precipitated proteins were washed extensively with ice-cold acetone. The acetone-washed pellets were then subjected to trypsin digestion, followed by methyl iodide cleavage. Released 3H-labeled isoprenoids were then extracted and resolved by C18 reverse-phase HPLC. Ki-Ras4B was cloned into vector pHIROI byBamHI sites to obtain the resulting GFP-Ki-Ras fusion construct (43Yokoe H. Meyer T. Nat. Biotech. 1996; 14: 1252-1256Crossref PubMed Scopus (165) Google Scholar). Ha-Ras was cloned into vector pHIRO2 byBamHI and XbaI cloning sites. pHIRO2 was constructed using pHIRO1 as a template by introducing a S65T mutation by polymerase chain reaction mutagenesis. The orientation of Ki-Ras and Ha-Ras and the integrity of the reading frame were verified by restriction analysis and sequencing of the GFP-Ras fusion constructs.In vitro transcription and RNA processing of these fusion constructs were performed according to the procedure described in Ref.43Yokoe H. Meyer T. Nat. Biotech. 1996; 14: 1252-1256Crossref PubMed Scopus (165) Google" @default.
• W1985753931 created "2016-06-24" @default.
• W1985753931 creator A5009620598 @default.
• W1985753931 creator A5010834737 @default.
• W1985753931 creator A5021268165 @default.
• W1985753931 creator A5029329845 @default.
• W1985753931 creator A5047333998 @default.
• W1985753931 date "1997-11-01" @default.
• W1985753931 modified "2023-10-16" @default.
• W1985753931 title "Prenylation-dependent Association of Ki-Ras with Microtubules" @default.
• W1985753931 cites W127937226 @default.
• W1985753931 cites W1485403581 @default.
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