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• W2003519013 abstract "Rho GTPases (20 human members) comprise a major branch of the Ras superfamily of small GTPases, and aberrant Rho GTPase function has been implicated in oncogenesis and other human diseases. Although many of our current concepts of Rho GTPases are based on the three classical members (RhoA, Rac1, and Cdc42), recent studies have revealed the diversity of biological functions mediated by other family members. A key basis for the functional diversity of Rho GTPases is their association with distinct subcellular compartments, which is dictated in part by three posttranslational modifications signaled by their carboxyl-terminal CAAX (where C represents cysteine, A is an aliphatic amino acid, and X is a terminal amino acid) tetrapeptide motifs. CAAX motifs are substrates for the prenyltransferase-catalyzed addition of either farnesyl or geranylgeranyl isoprenoid lipids, Rce1-catalyzed endoproteolytic cleavage of the AAX amino acids, and Icmt-catalyzed carboxyl methylation of the isoprenylcysteine. We utilized pharmacologic, biochemical, and genetic approaches to determine the sequence requirements and roles of CAAX signal modifications in dictating the subcellular locations and functions of the Rho GTPase family. Although the classical Rho GTPases are modified by geranylgeranylation, we found that a majority of the other Rho GTPases are substrates for farnesyltransferase. We found that the membrane association and/or function of Rho GTPases are differentially dependent on Rce1- and Icmt-mediated modifications. Our results further delineate the sequence requirements for prenyltransferase specificity and functional roles for protein prenylation in Rho GTPase function. We conclude that a majority of Rho GTPases are targets for pharmacologic inhibitors of farnesyltransferase, Rce1, and Icmt. Rho GTPases (20 human members) comprise a major branch of the Ras superfamily of small GTPases, and aberrant Rho GTPase function has been implicated in oncogenesis and other human diseases. Although many of our current concepts of Rho GTPases are based on the three classical members (RhoA, Rac1, and Cdc42), recent studies have revealed the diversity of biological functions mediated by other family members. A key basis for the functional diversity of Rho GTPases is their association with distinct subcellular compartments, which is dictated in part by three posttranslational modifications signaled by their carboxyl-terminal CAAX (where C represents cysteine, A is an aliphatic amino acid, and X is a terminal amino acid) tetrapeptide motifs. CAAX motifs are substrates for the prenyltransferase-catalyzed addition of either farnesyl or geranylgeranyl isoprenoid lipids, Rce1-catalyzed endoproteolytic cleavage of the AAX amino acids, and Icmt-catalyzed carboxyl methylation of the isoprenylcysteine. We utilized pharmacologic, biochemical, and genetic approaches to determine the sequence requirements and roles of CAAX signal modifications in dictating the subcellular locations and functions of the Rho GTPase family. Although the classical Rho GTPases are modified by geranylgeranylation, we found that a majority of the other Rho GTPases are substrates for farnesyltransferase. We found that the membrane association and/or function of Rho GTPases are differentially dependent on Rce1- and Icmt-mediated modifications. Our results further delineate the sequence requirements for prenyltransferase specificity and functional roles for protein prenylation in Rho GTPase function. We conclude that a majority of Rho GTPases are targets for pharmacologic inhibitors of farnesyltransferase, Rce1, and Icmt. Rho proteins are members of the Ras superfamily of small GTPases and function as GDP/GTP-regulated switches (1Colicelli J. Science's STKE 2004. 2004; : RE13Google Scholar, 2Wennerberg K. Rossman K.L. Der C.J. J. Cell Sci. 2005; 118: 843-846Crossref PubMed Scopus (986) Google Scholar). Much of our current understanding of the biochemistry and biology of the Rho family has come from the extensive evaluation of three classical members, RhoA, Rac1, and Cdc42 (3Wennerberg K. Der C.J. J. Cell Sci. 2004; 117: 1301-1312Crossref PubMed Scopus (469) Google Scholar). Similar to Ras, Rho GDP/GTP cycling is regulated by guanine nucleotide exchange factors that promote the formation of the active GTP-bound form (4Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (974) Google Scholar) and GTPase-activating proteins that catalyze the intrinsic GTPase activity and promote the formation of inactive GDP-bound Rho (5Bernards A. Settleman J. Trends Cell Biol. 2004; 14: 377-385Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Active, GTP-bound Rho GTPases bind preferentially to downstream effectors, stimulating diverse cytoplasmic signaling cascades that control actin reorganization and regulate cell shape, polarity, motility, adhesion, and membrane trafficking (6Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3794) Google Scholar). As such, it is thought that activated Rho proteins contribute to cancer progression by influencing the ability of cells to migrate and thus to invade and metastasize. In addition to these alterations in cellular function, aberrant activation of Rho proteins has also been shown to contribute to other cancer phenotypes by promoting cell growth, proliferation, survival, and angiogenesis (7Ridley A.J. Breast Cancer Res. Treat. 2004; 84: 13-19Crossref PubMed Scopus (156) Google Scholar). Therefore, defining pharmacologic approaches for inhibition of Rho GTPase function represents an important direction for target-based anti-cancer drug discovery.Similar to Ras, the majority of Rho family GTPases are known or anticipated to undergo a series of posttranslational modifications that promote proper subcellular localization to the plasma membrane and/or endomembranes, which is required for biological activity. This series of modifications is initiated by the recognition of a carboxyl-terminal CAAX tetrapeptide motif (where C represents cysteine, A is an aliphatic amino acid, and X is any amino acid), which is found on 16 of 20 Rho GTPases (Table 1; canonical CAAX motifs are not present in the Wrch-1, Chp/Wrch-2, RhoBTB1, or RhoBTB2). The first step, mediated by farnesyltransferase (FTase) 2The abbreviations used are: FTasefarnesyltransferaseRce1Ras-converting enzyme 1Icmtisoprenylcysteine-O-carboxyl methyltransferaseGGTase-Igeranylgeranyltransferase type IFTIfarnesyltransferase inhibitorGGTIgeranylgeranyltransferase I inhibitorMEFmouse embryonic fibroblastGFPgreen fluorescent protein2-BP2-bromopalmitateBiotin-BMCC1-biotinamido-4-(4′-(maleimidomethyl cyclohexanecarboxamido) butaneGTPaseguanine triphosphataseGFPgreen fluorescent protein. 2The abbreviations used are: FTasefarnesyltransferaseRce1Ras-converting enzyme 1Icmtisoprenylcysteine-O-carboxyl methyltransferaseGGTase-Igeranylgeranyltransferase type IFTIfarnesyltransferase inhibitorGGTIgeranylgeranyltransferase I inhibitorMEFmouse embryonic fibroblastGFPgreen fluorescent protein2-BP2-bromopalmitateBiotin-BMCC1-biotinamido-4-(4′-(maleimidomethyl cyclohexanecarboxamido) butaneGTPaseguanine triphosphataseGFPgreen fluorescent protein. and/or geranylgeranyltransferase type I (GGTase-I), results in the covalent addition of a farnesyl or geranylgeranyl isoprenoid lipid, respectively, to the cysteine residue of the CAAX sequence. Next, the -AAX peptide is cleaved from the carboxyl terminus by the Rce1 (Ras-converting enzyme 1) endoprotease. Finally, isoprenylcysteine-O-carboxyl methyltransferase (Icmt) catalyzes the addition of a methyl group to the prenylated cysteine residue (8Sebti S.M. Der C.J. Nat. Rev. Cancer. 2003; 3: 945-951Crossref PubMed Scopus (152) Google Scholar). Together, these modifications increase protein hydrophobicity and facilitate membrane association. Where studied, mutation of the cysteine residue of the CAAX motif, which prevents all three modifications, renders Rho GTPases inactive due to mislocalization to the cytosol (9Winter-Vann A.M. Casey P.J. Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (274) Google Scholar). Thus, pharmacological inhibitors of protein prenylation are anticipated to be effective inhibitors of Rho GTPase activity. Recent observations upon genetic ablation of GGTase-I activity support this possibility. Transient genetic depletion of GGTase-I caused mouse embryonic fibroblasts to undergo growth arrest, cell rounding, impaired cell migration, and reduced actin polymerization, and these phenotypic alterations were partially rescued by GGTase-I-independent, farnesylated variants of RhoA and Cdc42 (10Sjogren A.K. Andersson K.M. Liu M. Cutts B.A. Karlsson C. Wahlstrom A.M. Dalin M. Weinbaum C. Casey P.J. Tarkowski A. Swolin B. Young S.G. Bergo M.O. J. Clin. Invest. 2007; 117: 1294-1304Crossref PubMed Scopus (93) Google Scholar). These phenotypic consequences are consistent with loss of Rho GTPase function but additionally suggest that multiple GGTase-I substrates are important for regulation of cell morphology and actin organization. Similarly, loss of GGTase-I activity was lethal in the budding yeast Saccharomyces cerevisiae, and the combined expression of GGTase-I-independent, farnesylated variants of RhoA and Cdc42 suppressed this lethality (11Ohya Y. Qadota H. Anraku Y. Pringle J.R. Botstein D. Mol. Biol. Cell. 1993; 4: 1017-1025Crossref PubMed Scopus (57) Google Scholar).TABLE 1Carboxyl-terminal membrane-targeting sequence elements of human Ras and Rho GTPasesa Polybasic residues are in boldface type; putative or known palmitoylated (palm) cysteines are shaded gray; known or putative CAAX prenylation motifs are in boldface type and underlined; underlined sequences comprise a GDP/GTP binding motif.b Compiled from references cited in Ref. 67Ridley A.J. Trends Cell Biol. 2006; 16: 522-529Abstract Full Text Full Text PDF PubMed Scopus (863) Google Scholar; RhoG and RhoH (68Aspenstrom P. Fransson A. Saras J. Biochem. J. 2004; 377: 327-337Crossref PubMed Scopus (305) Google Scholar); Wrch-1 (23Berzat A.C. Buss J.E. Chenette E.J. Weinbaum C.A. Shutes A. Der C.J. Minden A. Cox A.D. J. Biol. Chem. 2005; 280: 33055-33065Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar); Chp (28Chenette E.J. Abo A. Der C.J. J. Biol. Chem. 2005; 280: 13784-13792Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). PM, plasma membrane; ER, endoplasmic reticulum; NE, nuclear envelope; MT, mitochondria.c The GTPase domains are followed by carboxyl-terminal tandem BTB domains. Open table in a new tab Although the CAAX-signaled posttranslational modifications are necessary for Ras and Rho GTPase function and membrane association, these three modifications alone are not sufficient to promote full membrane association or to target the proteins to the specific cellular subdomains required for proper GTPase function (12Cox A.D. Der C.J. Curr. Opin. Cell Biol. 1992; 4: 1008-1016Crossref PubMed Scopus (201) Google Scholar). Instead, at least two distinct sequence elements positioned immediately upstream of the CAAX motif serve as additional signals that are required to promote efficient membrane association and biological function. One element is composed of clusters of polybasic amino acid residues, as seen in K-Ras4B, that provide a positive charge that facilitates association with acidic membrane-associated lipids. The second sequence element present upstream of CAAX in some Rho GTPases is one or two cysteine residues that undergo post-translational modification by the fatty acid palmitate. Palmitoylated cysteines comprise the additional targeting signal for H-Ras and N-Ras proteins as well as for some Rho family GTPases (RhoB and TC10). Mutant Ras proteins that undergo the CAAX-signaled modifications but lack either the polybasic residues or palmitoylated cysteine(s) are mislocalized and are significantly compromised in their biological activities. Finally, additional sequences flanking these elements form a largely uncharacterized third signal that also contributes to dictating the precise subcellular localization of Ras and Rho GTPases (13Willumsen B.M. Cox A.D. Solski P.A. Der C.J. Buss J.E. Oncogene. 1996; 13: 1901-1909PubMed Google Scholar, 14Parton R.G. Hancock J.F. Trends Cell Biol. 2004; 14: 141-147Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 15Quatela S.E. Philips M.R. Curr. Opin. Cell Biol. 2006; 18: 162-167Crossref PubMed Scopus (53) Google Scholar). These locations can vary significantly; whereas some Rho GTPases are found predominantly at the plasma membrane (e.g. Rac1), some are associated mainly with endomembranes (e.g. RhoH), and still others are associated with endosomes (e.g. RhoD) (Table 1).Because of the importance of CAAX-signaled modifications for small GTPase localization and function, farnesyltransferase inhibitors (FTIs) were developed initially as anti-Ras therapies for cancer treatment. Unfortunately, K-Ras and N-Ras (the two Ras isoforms most commonly mutated in human cancers) undergo alternative prenylation by GGTase-I when in the presence of FTIs and therefore escape FTI-mediated inhibition of membrane association (16Rowell C.A. Kowalczyk J.J. Lewis M.D. Garcia A.M. J. Biol. Chem. 1997; 272: 14093-14097Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 17Whyte D.B. Kirschmeier P. Hockenberry T.N. Nunez-Oliva I. James L. Catino J.J. Bishop W.R. Pai J.K. J. Biol. Chem. 1997; 272: 14459-14464Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar). Nevertheless, FTIs have exhibited anti-tumor activity in preclinical and clinical trial analyses, presumably due to the inhibition of function of other FTase substrates (8Sebti S.M. Der C.J. Nat. Rev. Cancer. 2003; 3: 945-951Crossref PubMed Scopus (152) Google Scholar). In light of the role of aberrant Rho GTPase function in oncogenesis, Rho family GTPases (e.g. RhoB) are logical candidates for key targets of FTIs (18Prendergast G.C. Curr. Opin. Cell Biol. 2000; 12: 166-173Crossref PubMed Scopus (131) Google Scholar). Although GGTase-I modifies the classical Rho GTPases, the nature of the CAAX sequences of other members suggests that they may be FTase substrates.The observation that K-Ras and N-Ras undergo alternative prenylation in response to FTI treatment has also stimulated interest in the development of inhibitors that block other enzymes that facilitate Ras membrane association. First, GGTase-I inhibitors (GGTIs) were developed to block the function of the alternatively prenylated Ras proteins (19Sebti S.M. Hamilton A.D. Oncogene. 2000; 19: 6584-6593Crossref PubMed Scopus (271) Google Scholar). Furthermore, with increasing evidence for the involvement of normally geranylgeranylated proteins in cancer (e.g. Ral and Rho GTPases) (7Ridley A.J. Breast Cancer Res. Treat. 2004; 84: 13-19Crossref PubMed Scopus (156) Google Scholar, 20Sahai E. Marshall C.J. Nat. Rev. Cancer. 2002; 2: 133-142Crossref PubMed Scopus (1215) Google Scholar), there is now additional interest in the development of GGTIs to target these GGTase-I substrates for cancer treatment. Second, efforts to develop inhibitors of Rce1 and Icmt as novel anti-cancer agents have recently intensified (9Winter-Vann A.M. Casey P.J. Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (274) Google Scholar). However, there is concern regarding their effectiveness, since Ras proteins that fail to undergo these two modifications do retain partial localization and function (21Kato 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 (552) Google Scholar, 22Michaelson D. Ali W. Chiu V.K. Bergo M. Silletti J. Wright L. Young S.G. Philips M. Mol. Biol. Cell. 2005; 16: 1606-1616Crossref PubMed Scopus (127) Google Scholar). Additionally, since many FTase and GGTase-I substrates are also substrates for these two enzymes, there is also concern that such inhibitors will affect a broad array of cellular proteins and cause significant cell toxicity in normal cells. Support for this latter concern is provided by the observed embryonic lethality in mice deficient in either Rce1 or Icmt. Whether similar toxicity would be seen in adult animals is an important area of investigation.In light of the essential function of Rho family GTPases in normal cell physiology and their aberrant activation in oncogenesis (7Ridley A.J. Breast Cancer Res. Treat. 2004; 84: 13-19Crossref PubMed Scopus (156) Google Scholar, 20Sahai E. Marshall C.J. Nat. Rev. Cancer. 2002; 2: 133-142Crossref PubMed Scopus (1215) Google Scholar), establishing the sensitivity of Rho GTPases to FTI and GGTI inhibitors and the contribution of Rce1- and Icmt-catalyzed modifications to their cellular functions will be critical to the successful development of inhibitors of CAAX-signaled modifications. Therefore, we have utilized pharmacologic and genetic approaches to establish the importance of CAAX-signaled modifications for the functions of the less studied Rho GTPases. We found that, in contrast to the common perception based on the study of the classical Rho GTPases, farnesylation is a lipid modification that is equally important as geranylgeranylation for Rho GTPase function. Furthermore, we conclude that the rules governing palmitoylation of cysteine-containing signal sequences and even CAAX tetrapeptide prenyltransferase specificity derived from structural studies are imprecise and that experimental analyses are still required to establish the lipid modification status of a particular CAAX-terminating protein. Finally, our observations that Rho GTPase subcellular localization and/or function depend on Rce1 and/or Icmt enzymatic activity support the value of developing inhibitors of these two enzymes as therapeutic strategies to block Rho GTPase function.EXPERIMENTAL PROCEDURESExpression Constructs and Cell Culture Lipid Inhibitor Analyses—cDNAs for human Ras and Rho GTPases (H-Ras, K-Ras, RhoA, Rnd1, and Rnd2 and Rnd3, RhoD, RhoH, TC10, TCL, and Rif) and rat RhoB were cloned into pEGFP mammalian expression vectors (Clontech) as previously described (23Berzat A.C. Buss J.E. Chenette E.J. Weinbaum C.A. Shutes A. Der C.J. Minden A. Cox A.D. J. Biol. Chem. 2005; 280: 33055-33065Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 24Michaelson D. Silletti J. Murphy G. D'Eustachio P. Rush M. Philips M.R. J. Cell Biol. 2001; 152: 111-126Crossref PubMed Scopus (558) Google Scholar) or constructed for this study. All constructs were sequence-verified, and cloning details are available upon request.HEK 293T cells were maintained in Dulbecco's modified minimum essential medium supplemented with 10% fetal calf serum (Sigma), 100 units/ml penicillin, and 100 μg/ml streptomycin. NIH 3T3 cells were maintained in Dulbecco's modified minimum essential medium supplemented with 10% calf serum (Sigma) and 100 units/ml penicillin and 100 μg/ml streptomycin (“complete growth medium”). Spontaneously immortalized mouse embryonic fibroblasts (MEFs) were originally prepared from Icmt–/– and Rce1–/– mouse embryos, along with control fibroblasts (Icmt+/+ and Rce1+/+) from littermate embryos (25Bergo M.O. Leung G.K. Ambroziak P. Otto J.C. Casey P.J. Gomes A.Q. Seabra M.C. Young S.G. J. Biol. Chem. 2001; 276: 5841-5845Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and were kindly provided by Stephen G. Young (UCLA, Los Angeles, CA). MEF cultures were maintained in Dulbecco's modified minimum essential medium supplemented with 15% calf serum (Colorado Serum, Denver, CO), nonessential amino acids, and l-glutamine.The highly selective inhibitors of FTase (FTI-2153) and of GGTase-I (GGTI-2417) were provided by Saïd Sebti (Moffitt Cancer Center) and Andrew Hamilton (Yale) and were dissolved in DMSO (26Falsetti S.C. Wang D.A. Peng H. Carrico D. Cox A.D. Der C.J. Hamilton A.D. Sebti S.M. Mol. Cell Biol. 2007; 27: 8003-8014Crossref PubMed Scopus (65) Google Scholar, 27Sun J. Blaskovich M.A. Knowles D. Qian Y. Ohkanda J. Bailey R.D. Hamilton A.D. Sebti S.M. Cancer Res. 1999; 59: 4919-4926PubMed Google Scholar). The palmitate analog 2-bromopalmitate (2-BP), an inhibitor widely used to evaluate the role of protein palmitoylation in protein targeting (28Chenette E.J. Abo A. Der C.J. J. Biol. Chem. 2005; 280: 13784-13792Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 29Veit M. Laage R. Dietrich L. Wang L. Ungermann C. EMBO J. 2001; 20: 3145-3155Crossref PubMed Scopus (77) Google Scholar, 30Webb Y. Hermida-Matsumoto L. Resh M.D. J. Biol. Chem. 2000; 275: 261-270Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar), was purchased from Sigma and dissolved in ethanol. Control cultures were treated with the equivalent final concentration of ethanol or DMSO (designated vehicle). In the inhibitor assays, cells were transfected as described below, washed, and incubated for 20 h with growth medium supplemented with 10 μm FTI-2153, 10 μm GGTI-2417, or 100 μm 2-BP.Transfection, Immunofluorescence, and Microscopy—For live cell microscopy, cells were plated, transfected, and imaged in a 35-mm culture dish that incorporated a number 1.5 glass coverslip-sealed 15-mm cut-out on the bottom (MatTek, Ashland, MA). Uncoated dishes were used for NIH 3T3 cells, and poly-d-lysine-coated dishes were used for all experiments using MEFs. DNA transfections were performed with Lipofectamine Plus reagent according to the manufacturer's instructions (Invitrogen). Three h after transfection, cells were washed, grown in phenol red-free Dulbecco's modified minimum essential medium/F-12 supplemented with 10% calf serum, and treated with inhibitors where indicated.For immunofluorescence, cells transiently transfected with plasmid DNAs encoding GFP fusion constructs of small GTPases were fixed 24 h after transfection with 4% paraformaldehyde, permeabilized with Triton X-100, stained with Alexa 594-phalloidin (Molecular Probes, Inc., Eugene, OR), and mounted with FluorSave (Calbiochem).For both live cell imaging and immunofluorescence studies, cells were examined with an inverted laser-scanning confocal microscope (Zeiss 510 LSM) using an oil immersion ×63 numerical aperture 1.4 objective. Images were captured by scanning with the 488 nm spectral line of an argon-ion laser using the LP 505 emission filter (for live cell imaging; GFP) or sequential scanning with the 488 nm argon laser and the 543 nm HeNe1 laser and the BP 505–530 (for GFP) or LP 585 (for Alexa 594) emission filters. 0.3-μm confocal z-sections that show both nuclear and membrane/cytosolic localization of GFP fusion proteins were obtained and analyzed. Brightness and contrast of JPEG images were adjusted using Adobe Photoshop CS2 software.Transformation Assays—For soft agar colony formation analyses of anchorage-independent growth, NIH 3T3 cells stably expressing activated Rac or Rac C178S mutants were seeded at a density of 105 cells/60-mm dish in a solution of complete growth medium containing 0.4% bacto-agar over a layer of complete growth medium containing 0.6% bacto-agar. Colonies were allowed to form for 2 weeks, after which viable colonies were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium salt. Plates were scanned, and the number of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-positive colonies was quantified using ImageJ software. Results for transformation assays are representative of at least three experiments from independently generated sets of stable cell lines.1-Biotinamido-4-(4′-(maleimidomethyl Cyclohexanecarboxamido) Butane (Biotin-BMCC) Labeling—Analyses of protein palmitoylation were done as described in Refs. 31Drisdel R.C. Green W.N. BioTechniques. 2004; 36: 276-285Crossref PubMed Google Scholar and 32Shutes A. Berzat A.C. Chenette E.J. Cox A.D. Der C.J. Methods Enzymol. 2006; 406: 11-26Crossref PubMed Scopus (20) Google Scholar). Briefly, 293T cells were transfected with 7 μg of the indicated pEGFP construct using a calcium phosphate transfection technique. Forty-eight h after transfection, cells were lysed and incubated with 5 μg of anti-GFP monoclonal antibody (JL-8; Clontech) at 4 °C for 1 h, at which point 20 μg of protein G (Invitrogen) was added to the lysates and incubated at 4 °C for 1 h. Bound protein was washed and incubated with lysis buffer containing 50 mm N-ethylmaleimide (Sigma) for 48 h at 4 °C. Bound protein was then washed and treated with 1 m hydroxylamine, pH 7.4, to cleave thioester bonds for 1 h at 25 °C, washed again, and treated with biotin-BMCC (Pierce), which recognizes free sulfhydryl groups, for 2 h at 25 °C. Bound protein was washed again, resuspended in 50 μl of 2 × sample loading buffer, resolved by 12% SDS-PAGE, and transferred to polyvinylidene difluoride membrane. Labeled protein was detected by incubation with streptavidin-horseradish peroxidase (Pierce), and the membrane was washed and exposed to x-ray film. Twenty μg of lysate was resolved by SDS-PAGE, transferred, incubated with anti-GFP primary antibody and anti-mouse IgG-horseradish peroxidase secondary antibody, and exposed to x-ray film to verify the presence of each GFP-tagged protein.In Vitro Prenylation Analyses—Full-length human Rnd, RhoB, and TC10 proteins were expressed as fusion proteins containing six histidine residues at the amino terminus. The cDNA coding regions were amplified from appropriate cell lines by PCR and subcloned into the pQE bacterial expression vector (Qiagen). The identity of all plasmids was confirmed by restriction mapping and DNA sequencing of the PCR-amplified fragments. Six-histidine-tagged Rnd1, Rnd2, Rnd3, and TC10 were expressed and purified from Escherichia coli by nickel affinity chromatography, as we have described previously for Ras proteins (33Zhang F.L. Kirschmeier P. Carr D. James L. Bond R.W. Wang L. Patton R. Windsor W.T. Syto R. Zhang R. Bishop W.R. J. Biol. Chem. 1997; 272: 10232-10239Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Expression and purification of recombinant human FTase and GGTase-I from SF9 insect cells (∼50% pure) were performed as we have described elsewhere (33Zhang F.L. Kirschmeier P. Carr D. James L. Bond R.W. Wang L. Patton R. Windsor W.T. Syto R. Zhang R. Bishop W.R. J. Biol. Chem. 1997; 272: 10232-10239Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). FTase and GGTase-I activity were determined by measuring the transfer of [3H]farnesyl or [3H]geranylgeranyl to the Rho GTPase substrate in reaction mixtures containing (in 200 μl) 50 mm Tris·HCl (pH 7.5), 1 mm dithiothreitol, 20 mm KCl, 5 mm MgCl2. The concentration of recombinant FTase and GGTase-I was 0.5 μm, whereas the GTPase protein substrate concentrations were varied from 0 to 1.0 μm. Reactions were started by the addition of 20 ng of FTase or GGTase-I and proceeded for 4 min at 37 °C.Statistical Analysis—Data were analyzed by the use of Student's t test. In all analyses, p < 0.05 was considered statistically significant, and data are presented as mean ± S.D.RESULTSInhibition of Farnesyltransferase Blocks Rho Family GTPase Localization and Function—Substrate specificity of FTase and GGTase-I toward small GTPases is determined primarily by the sequence of the CAAX tetrapeptide motif (Table 1). Biochemical and structural studies of CAAX peptides in complex with FTase and GGTase-I have defined rules that govern substrate selectivity (34Reid T.S. Terry K.L. Casey P.J. Beese L.S. J. Mol. Biol. 2004; 343: 417-433Crossref PubMed Scopus (211) Google Scholar). Whereas the specific isoprenoid modification of the classical Rho GTPases and of their highly related isoforms (RhoA/B/C, Rac1/2/3, and Cdc42) has been confirmed in vivo, the precise isoprenoid modification of the majority of Rho family GTPases has not been tested. Where studied, the prenylation of Ras and Rho small GTPases has been found to be essential for proper subcellular localization. Therefore, to determine the importance of farnesylation or geranylgeranylation to Rho GTPase localization, we ectopically expressed GFP-fusion Rho GTPase proteins to visualize their subcellular localization in live cells and determined the ability of treatment with the potent and highly selective FTI (FTI-2153) and/or GGTI (GGTI-2417) to alter their subcellular location (35Berzat A.C. Brady D.C. Fiordalisi J.J. Cox A.D. Methods Enzymol. 2005; 407: 575-597Crossref Scopus (26) Google Scholar). Exogenous expression of GFP fusion proteins has been used extensively to evaluate the subcellular localization of small GTPases and has been validated as an accurate reflection of the subcellular location of the endogenous protein (24Michaelson D. Silletti J. Murphy G. D'Eustachio P. Rush M. Philips M.R. J. Cell Biol. 2001; 152: 111-126Crossref PubMed Scopus (558) Google Scholar, 36Choy E. Chiu V.K. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I.E. Philips M.R. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar, 37Heo W.D. Meyer T. Cell. 2003; 113: 315-328Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). For these analyses, we utilized wild-type (and therefore predominantly GDP-bound) human Rho GTPases to avoid potential complications in Rho GTPase localization due to effector association or to altered interaction with Rho GDP dissociation inhibitor proteins seen with the activated forms of some Rho GTPases (24Michaelson D. Silletti J. Murphy G. D'Eustachio P. Rush M. Philips M.R. J. Cell Biol. 2001; 152: 111-126Crossref PubMed Scopus (558) Google Scholar, 36Choy E. Chiu V.K. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I.E. Philips M.R. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar, 37Heo W.D." @default.
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• W2003519013 date "2008-09-01" @default.
• W2003519013 modified "2023-10-10" @default.
• W2003519013 title "Rho Family GTPase Modification and Dependence on CAAX Motif-signaled Posttranslational Modification" @default.
• W2003519013 cites W1487258982 @default.
• W2003519013 cites W1492170959 @default.
• W2003519013 cites W1503951074 @default.
• W2003519013 cites W1517546728 @default.
• W2003519013 cites W1577261854 @default.
• W2003519013 cites W1583605194 @default.
• W2003519013 cites W1839381104 @default.
• W2003519013 cites W1968453135 @default.
• W2003519013 cites W1970606222 @default.
• W2003519013 cites W1974624332 @default.
• W2003519013 cites W1975702431 @default.
• W2003519013 cites W1986587815 @default.
• W2003519013 cites W1990689067 @default.
• W2003519013 cites W1993069357 @default.
• W2003519013 cites W1993418815 @default.
• W2003519013 cites W1995066828 @default.
• W2003519013 cites W1996174856 @default.
• W2003519013 cites W2004799153 @default.
• W2003519013 cites W2006777514 @default.
• W2003519013 cites W2010207338 @default.
• W2003519013 cites W2012932684 @default.
• W2003519013 cites W2014018734 @default.
• W2003519013 cites W2017098460 @default.
• W2003519013 cites W2017131142 @default.
• W2003519013 cites W2019731805 @default.
• W2003519013 cites W2019785646 @default.
• W2003519013 cites W2020075607 @default.
• W2003519013 cites W2022076650 @default.
• W2003519013 cites W2030911680 @default.
• W2003519013 cites W2041953777 @default.
• W2003519013 cites W2051626784 @default.
• W2003519013 cites W2052715806 @default.
• W2003519013 cites W2054365538 @default.
• W2003519013 cites W2058569850 @default.
• W2003519013 cites W2083506343 @default.
• W2003519013 cites W2085707729 @default.
• W2003519013 cites W2086606183 @default.
• W2003519013 cites W2087197236 @default.
• W2003519013 cites W2090356463 @default.
• W2003519013 cites W2094993800 @default.
• W2003519013 cites W2095562167 @default.
• W2003519013 cites W2097032963 @default.
• W2003519013 cites W2102492191 @default.
• W2003519013 cites W2107272456 @default.
• W2003519013 cites W2112078762 @default.
• W2003519013 cites W2131376017 @default.
• W2003519013 cites W2137566722 @default.
• W2003519013 cites W2142475134 @default.
• W2003519013 cites W2143535564 @default.
• W2003519013 cites W2148271712 @default.
• W2003519013 cites W2149189973 @default.
• W2003519013 cites W2152401699 @default.
• W2003519013 cites W2154655439 @default.
• W2003519013 cites W2156654096 @default.
• W2003519013 cites W2165803049 @default.
• W2003519013 cites W2166867865 @default.
• W2003519013 cites W2167158605 @default.
• W2003519013 cites W2169336337 @default.
• W2003519013 cites W2254098459 @default.
• W2003519013 cites W2312397356 @default.
• W2003519013 cites W35736013 @default.
• W2003519013 cites W4232023895 @default.
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