Disruption of the Mouse Rce1 Gene Results in Defective Ras Processing and Mislocalization of Ras within Cells*

Edward KimDagger §parallel , Patricia AmbroziakDagger , James C. Otto**Dagger Dagger , Brigit Taylor§§, Matthew Ashby¶¶, Kevin Shannon§§, Patrick J. Casey**, and Stephen G. YoungDagger §

From the Dagger  Gladstone Institute of Cardiovascular Disease, § Cardiovascular Research Institute, Departments of  Medicine and §§ Pediatrics, University of California, San Francisco, California 94141-9100, ¶¶ Acacia Biosciences, Inc., Richmond, California 94806, and the ** Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710-3686

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Little is known about the enzyme(s) required for the endoproteolytic processing of mammalian Ras proteins. We identified a mouse gene (designated Rce1) that shares sequence homology with a yeast gene (RCE1) implicated in the proteolytic processing of Ras2p. To define the role of Rce1 in mammalian Ras processing, we generated and analyzed Rce1-deficient mice. Rce1 deficiency was lethal late in embryonic development (after embryonic day 15.5). Multiple lines of evidence revealed that Rce1-deficient embryos and cells lacked the ability to endoproteolytically process Ras proteins. First, Ras proteins from Rce1-deficient cells migrated more slowly on SDS-polyacrylamide gels than Ras proteins from wild-type embryos and fibroblasts. Second, metabolic labeling of Rce1-deficient cells revealed that the Ras proteins were not carboxymethylated. Finally, membranes from Rce1-deficient fibroblasts lacked the capacity to proteolytically process farnesylated Ha-Ras, N-Ras, and Ki-Ras or geranylgeranylated Ki-Ras. The processing of two other prenylated proteins, the farnesylated Ggamma 1 subunit of transducin and geranylgeranylated Rap1B, was also blocked. The absence of endoproteolytic processing and carboxymethylation caused Ras proteins to be mislocalized within cells. These studies indicate that Rce1 is responsible for the endoproteolytic processing of the Ras proteins in mammals and suggest a broad role for this gene in processing other prenylated CAAX proteins.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Post-translational modifications are essential for the membrane targeting of Ras proteins and many other proteins containing a "CAAX sequence" at their C terminus (1-4). CAAX proteins terminate with the amino acids -C-A-A-X, where the "C" is a cysteine, the two "A" residues are usually aliphatic amino acids, and the "X" can be one of several amino acids. CAAX proteins undergo three sequential enzymatic post-translational modifications. First, either a farnesyl or geranylgeranyl isoprenoid lipid is attached to the thiol group of the cysteine residue by cytosolic protein prenyltransferases (5). In general, the cysteine is geranylgeranylated when the X residue is a leucine or a phenylalanine; otherwise, the protein is farnesylated (6, 7). Second, the last three amino acids of the protein (i.e. the -AAX) are removed by a specific endoprotease; this step is prenylation-dependent and is thought to take place on the cytoplasmic surface of the endoplasmic reticulum (8). Finally, the C group of the prenylcysteine is methylated by an endoplasmic reticulum-associated methyltransferase (1, 9, 10). In addition to these three processing events, some CAAX proteins, including Ha-Ras, Ki-Ras4A, and N-Ras, can be further modified by palmitoylation of upstream cysteine residues (11).

The mammalian prenyltransferase enzymes responsible for farnesylation and geranylgeranylation of the CAAX proteins have been defined (2, 5, 12), and a mammalian enzyme responsible for methylating the prenylcysteine of CAAX proteins was identified recently (13). However, progress in defining the mammalian enzymes for the middle step of the triad, the endoproteolysis step, has been limited. Enzymatic activities capable of carrying out the endoproteolysis step have long been recognized (10, 14-18). However, only very recently have specific molecules been linked to that step, and virtually all of this progress has occurred in yeast. Two genes from Saccharomyces cerevisiae, RCE1 and AFC1, have been shown to be involved in the proteolytic processing of several yeast CAAX proteins (19, 20). Rce1p (for Ras and a-factor-converting enzyme) is essential for cleaving the C-terminal three amino acids of a yeast Ras protein, Ras2p, and is active in processing the yeast mating pheromone a-factor (19, 20). Afc1p (for a-factor-converting enzyme, also described as Ste24p; Ref. 21) carries out two successive proteolytic processing steps for the precursor to a-factor: cleavage of the C-terminal three amino acids from the farnesylated precursor and the subsequent cleavage of 7 amino acids from the N-terminal portion of the protein (20-22). Afc1p does not process yeast Ras2p (19), nor does it process human farnesyl-Ki-Ras in an in vitro assay (8).

For several years, it has been clear that the prenylation of CAAX proteins, particularly the Ras proteins, is critical for plasma membrane targeting and for biological function (11, 23). However, the functional importance of the proteolysis and methylation steps has been less obvious. Those "post-prenylation" steps may simply render prenylated proteins more hydrophobic and thereby enhance their membrane affinity (24, 25). It has also been suggested that those processing steps could enhance the stability of some prenylated proteins (26). In any case, several studies have suggested that the post-prenylation processing steps might be important for the function of the Ras proteins. For example, a mutant Ki-Ras4B that was prenylated but not further processed exhibited reduced transforming activity when expressed in 3T3 fibroblasts (27). Moreover, in RCE1-deficient yeast (rce1Delta ), Ras2p was mislocalized to the cytosol and internal membranes, and the yeast were protected from the heat sensitivity elicited by the expression of a mutationally activated Ras2p. Interestingly, rce1Delta yeast grew as well as wild-type cells under normal conditions, despite the defect in Ras processing (19). The fact that blocking Rce1p-mediated CAAX protein proteolysis appeared to attenuate the biological effects of an activated yeast Ras protein without affecting the growth of yeast under normal conditions has led to speculation that inhibiting the proteolysis step might be a promising therapeutic strategy for treating human cancers caused by mutationally activated Ras proteins (19, 28).

The purpose of this study was to define the mammalian enzyme responsible for Ras endoproteolytic processing and to gain insights into the physiologic consequences of blocking that processing step. At the time that the RCE1 gene was identified in yeast, expressed sequence tags (ESTs)1 containing sequence homology to that gene existed within the mouse EST data bases. To determine whether these ESTs represented a bona fide functional homologue of the yeast gene with a role in the processing of mammalian Ras proteins, the mouse ESTs were used to identify a large genomic clone spanning the mouse gene (designated Rce1), which was then disrupted by gene targeting in mouse embryonic stem (ES) cells. Here, we describe the phenotype of Rce1-deficient mice and provide evidence that Rce1 is essential for the proteolytic processing of the mammalian Ras proteins. We also show that Rce1-deficient cells are defective in the proteolytic cleavage of other prenylated proteins, establishing a broader role for the product of the Rce1 gene in the processing of CAAX proteins.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Mouse Rce1 ESTs and a Mouse Rce1 Genomic Clone-- An XREFdb query of GenBankTM sequences with the yeast RCE1 coding sequences identified two mouse EST matches: I.M.A.G.E. clones 331228 (0.8 kb) and 608689 (1.1 kb) (Research Genetics, Huntsville, AL). These ESTs shared 71% amino acid homology and 52% identity with several portions of the yeast RCE1 gene in a region spanning amino acids 147-257. DNA sequencing and restriction mapping revealed that the mouse ESTs contained overlapping sequences spanning the majority of the mouse Rce1 cDNA. Screening of a bacterial artificial chromosome library of strain 129/Sv mouse genomic DNA (Research Genetics) with the 1.1-kb EST identified a clone (427 J 4) that spanned the mouse Rce1 gene.

Analysis of the Tissue Distribution of Rce1 Gene Expression-- An RNA Master Blot (CLONTECH, Palo Alto, CA) was used to identify the tissues that expressed the Rce1 gene. The blot was probed with a 223-base pair Rce1 cDNA fragment (corresponding to amino acids 136-210), which was amplified from a mouse liver cDNA library (CLONTECH, Palo Alto, CA) with oligonucleotide primers 5'-TTGCCCTTGTGACCTGACAGATGG-3' and 5'-GAGTGGGCAGGTGAACACAGCAGG-3'. To assess the relative levels of Rce1 gene expression in different tissues, the blot was analyzed with a phosphorimager (Fuji Bio-Imaging Analyzer, BAS 1000 with MacBAS; Fuji Medical Systems, Stamford, CT).

Generation of Rce1-deficient Mice-- A sequence replacement gene-targeting vector was constructed from a 10.5-kb BamHI fragment of the bacterial artificial chromosome that spanned the Rce1 gene. The vector was constructed in the plasmid pKSloxPNT (29) (provided by Dr. A. Joyner), which contains polylinker restriction sites, a thymidine kinase gene (tk), and a neomycin resistance gene (neo) flanked by loxP sites. The 5' arm of the vector, 3.8 kb long, contained the promoter sequences of the Rce1 gene and all of the coding sequences located 5' to an Eco47III site within an exon of the Rce1 gene (corresponding to amino acid 115 of the protein); the 3' arm (on the 3' side of the neo) extended from an EcoRV site located within the last exon of the gene (within the 3'-untranslated sequences of the mRNA) to an EcoRI site located ~3.6 kb 3' to the gene. Thus, the neo replaced the Eco47III-EcoRV segment of the Rce1 gene, which codes for the C-terminal two-thirds of the protein (amino acids 115-329). This vector was electroporated into mouse ES cells, and targeted colonies were identified by Southern analysis of XbaI-cleaved genomic DNA with a 1-kb EcoRI-BamHI probe located 3' to the sequences in the gene-targeting vector. Two targeted ES cell clones were used to generate Rce1-deficient mice, according to published techniques (30). All mice described here had a mixed genetic background (~50% C57BL/6 and ~50% 129/Sv). The mice were weaned at 21 days of age, housed in a barrier facility with a 12-h light/dark cycle, and fed a chow diet containing 4.5% fat (Ralston Purina, St. Louis, MO).

Examination of Rce1-deficient Mouse Embryos-- Timed matings were established with Rce1+/- males and females as described previously (31). Pregnant female mice were euthanized between 10.5 and 20.5 days post coitus. After dissection of the embryo and fetal membranes from the uterus, embryos were fixed in 3% paraformaldehyde, and fetal membranes were used for genotyping. The embryos were sectioned, stained with hematoxylin and eosin, and examined by light microscopy.

Preparation of Fibroblasts from Rce1-deficient Mouse Embryos-- To isolate primary embryonic fibroblasts, Rce1+/- intercrosses were established, and pregnant females were sacrificed at 11.5 days post coitus. Fetal membranes were used for genotyping. The embryos were incubated in 5.0 ml of 0.25% trypsin-EDTA (Life Technologies, Inc.) for 8 h at 4 °C, followed by a 20-min incubation at 37 °C. After removal from the trypsin solution, the embryos were mechanically disrupted by repeated pipetting in 5 ml of Dulbecco's modified Eagle's medium supplemented with 10% v/v fetal bovine serum, L-glutamine, nonessential amino acids, penicillin-streptomycin (all from Life Technologies), and 2-mercaptoethanol (Sigma; final concentration, 100 µM). The debris was allowed to settle, and the cell suspension was removed, diluted to 25 ml with medium, and plated in T150 flasks. Approximately 1 × 107 cells were obtained from each embryo. The cells were grown in an incubator with 7% CO2 at 37 °C. To immortalize the fibroblasts, cells were passaged according to a 3T6 protocol (32), in which cells are harvested and counted every 3 days and replated at a density of 2 × 106 cells/100-mm dish.

Western Blot Analysis of Ras Proteins in Rce1-deficient Mice-- To prepare cell lysates of primary embryonic fibroblasts, confluent cells were harvested from a 100-mm plate in 0.5 ml of RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) and disrupted by sonication for 10 s in a Branson model 400 sonifier (duty cycle constant, output control 0.3; Danbury, CT). To obtain whole embryo lysates, E14.5 mouse embryos were isolated from pregnant Rce1+/- females that had been mated with a Rce1+/- male. The embryos were homogenized in 3.0 ml of phosphate-buffered saline (PBS; Digene Diagnostics, Beltsville, MD) containing 1% SDS and 50 mM 2-mercaptoethanol for 15 s with a Kinematica Polytron (Brinkman Instruments, Palo Alto, CA). The samples were then heated to 95 °C for 5 min and loaded onto an SDS/10-20% gradient polyacrylamide gel. After electrophoretic transfer of the proteins to a nitrocellulose membrane, Western blots performed with the "pan-Ras" monoclonal antibody Ab-4 (i.e. binds to N-Ras, Ki-Ras, and Ha-Ras; Oncogene Science, Uniondale, NY) or the Ki-Ras-specific monoclonal antibody Ab-1 (Oncogene Science), followed by an incubation with horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Pharmacia Biotech). Antibody binding was detected with the Enhanced Chemiluminescence kit (Amersham Pharmacia Biotech).

To immunoprecipitate the Ras proteins from fibroblasts or from the tissues of mouse embryos, lysates (200 µg of embryo lysate protein or the complete lysate from a 100-mm plate of confluent fibroblasts) were adjusted to 1.0 ml with RIPA buffer and pre-cleared by an incubation with protein A-Sepharose CL-4B beads (Sigma) or protein G-agarose beads (Boeringher Mannheim) for 30 min at 4 °C. The supernatant was then incubated with 5 µg of Y13-259 (Santa Cruz Biotechnology, Santa Cruz, CA), a pan-Ras-specific rat monoclonal antibody overnight at 4 °C. The immune complexes were then incubated with protein A- or protein G-agarose beads for 2 h at 4 °C and centrifuged for 5 min at 12,500 × g. After three washes with RIPA buffer, the pellets were resuspended in 20 µl of sample buffer. Immunoprecipitates were size-fractionated on an SDS/10-20% gradient polyacrylamide gel, and Western blots were performed with either antibody Ab-4 or Ab-1.

Methylation of Ras Proteins in Rce1-/- Fibroblasts-- To determine whether the C terminus of Ras was methylated in Rce1+/+ and Rce1-/- embryonic fibroblasts, we used a modification of the base-labile methylation assay described by Clarke et al. (1). Briefly, 100-mm dishes containing approximately 2.5 × 106 fibroblasts were incubated in the presence of 1 mCi of L-[methyl-3H]methionine (80 Ci/mmol; Amersham Pharmacia Biotech) and [35S]cysteine (>1000 Ci/mmol; Amersham Pharmacia Biotech) in methionine- and cysteine-free Dulbecco's modified Eagle's medium/10% fetal bovine serum in a 7% CO2 incubator for 24 h at 37 °C. The medium was removed, and the cells were washed with 5 ml of ice-cold PBS, scraped from the dish with a rubber policeman into 1 ml of ice-cold PBS, and centrifuged at 500 × g. Cell pellets were resuspended in 1.0 ml of RIPA lysis buffer and disrupted by sonification, as described above. Ras proteins were then immunoprecipitated with antibody Y13-259, and the immunoprecipitate was size-fractionated on an SDS/10-20% gradient polyacrylamide gel. The gel was fixed in isopropanol, water, and acetic acid (25:65:10, v/v), soaked for 30 min in Amplify (Amersham Pharmacia Biotech), and dried in a vacuum oven at 80 °C. The dried gel was imaged with a phosphorimager, and the area corresponding to the Ras band was excised and used to quantify the amount of [3H]methanol released by base hydrolysis of the cysteine methyl esters. The gel slice was placed in a 1.5-ml capless microcentrifuge tube, mixed with 200 µl of 2 M NaOH, and lowered into 20-ml scintillation vial containing 6 ml of Safety-Solve II counting fluor (RPI) fluid. The vial was then capped and incubated at 55 °C; within the capped vial, the [3H]methanol released from the cysteine methyl esters diffuse into the fluor, whereas the labeled protein remains in the gel slice. After 24 h, the microcentrifuge tubes were removed, and the radioactivity in the vials was counted.

CAAX Endoprotease Assay-- CAAX endoprotease activities were measured using a coupled assay. Recombinant prenyl proteins were first used as substrates in a proteolysis assay in which the protease source was membranes from either Rce1+/+ or Rce1-/- fibroblasts. The degree of specific proteolytic processing of the prenyl proteins was then determined by quantifying their ability to be methylated by the yeast prenyl protein carboxymethyltransferase STE14 produced in Sf9 cells.

Substrate proteins containing a CAAX sequence were expressed in Escherichia coli, prenylated in vitro with recombinant protein farnesyltransferase or protein geranylgeranyltransferase, and either purified (in the case of farnesyl-Ki-Ras and geranylgeranyl-Ki-Ras) or simply concentrated as a mixture of prenylated protein, nonprenylated protein, and the prenyltransferase (33). Ki-Ras, N-Ras, and Rap-1B were modified at the N terminus with a hexahistidine tag, whereas Ggamma 1 and Ha-Ras were unmodified. To prepare membranes from Rce1+/+ and Rce1-/- embryonic fibroblasts, cells were lysed by sonication. After centrifugation at 500 × g for 10 min to remove cellular debris and nuclei, the membrane fraction was ultracentrifuged at 200,000 × g for 1.5 h and resuspended in 50 mM Tris-HCl at protein concentrations of 10-25 mg/ml.

To assay for the prenyl protein-specific protease, prenyl protein substrates and membrane fractions were mixed in 50 µl of a 100 mM Hepes buffer, pH 7.5, containing 5 mM MgCl2. After a 30-min incubation at 37 °C, the proteolysis reactions were stopped, and the methyltransferase reactions were initiated by adding a mixture (20 µl) containing 5 mM NaHPO4, 87.5 mM EDTA, a variety of protease inhibitors (300 µM N-tosyl-L-phenylalanine chloromethylketone, 1 mM phenylmethylsulfonyl fluoride, 10 mM 1,10-phenanthroline), 20 µg of Sf9/STE14 membranes, and 17.5 µM [3H]S-adenosylmethionine (1.5 Ci/mmol). After a 20-min incubation at 37 °C, the methylation reaction was terminated by adding 0.5 ml of 4% SDS. Bovine brain cytosol (50 µg of protein) was added as a carrier protein, and the mixture was incubated for 20 min at room temperature, whereupon the proteins were precipitated by adding 0.5 ml of 30% trichloroacetic acid. Precipitated proteins were collected on glass fiber filters (for the Ras proteins and Rap1B) or nitrocellulose filters (for Ggamma 1), and the extent of proteolysis was determined by quantifying 3H incorporation by scintillation counting.

Subcellular Fractionation and Localization of Ras Proteins in Fibroblasts-- To determine the degree of membrane association of Ras, subcellular fractionation of primary cells and transformed cells was performed as described (34). For each sample, 100-mm dishes containing confluent fibroblasts were washed with ice-cold PBS, and cells were collected in 1.0 ml of PBS and centrifuged at 500 × g for 10 min. Cell pellets were swelled with 1225 µl of hypotonic buffer (10 mM Tris-HCl, pH 7.5, 1.0 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin, and 1 µM dithiothreitol) and placed on ice for 10 min. Cells were disrupted with an ice-cold Dounce tissue homogenizer, after which 225 µl of 1 M NaCl was added. A total of 450 µl of this solution ("total lysate") was transferred to a microfuge tube and set aside. The remaining 1000 µl was transferred to a polycarbonate ultracentrifuge tube and spun at 100,000 × g for 30 min at 4 °C. The supernatant fluid (S100, representing the cytosolic fraction) was transferred to a new microcentrifuge tube; the pellet (P100, representing the membrane fraction) was resuspended in 850 µl of hypotonic buffer and 150 µl of 1 M NaCl. Next, 50 µl of 10 × Hi-SDS RIPA buffer was added to total lysate sample, and 110 µl was added to the S100 and P100 fractions. After incubation on ice for 10 min, the lysates were clarified by centrifugation at 25,000 × g for 30 min at 4 °C. Supernatants were transferred to a new microfuge tube, and immunoprecipitations, gel fractionations, and Western blot detection of the Ras proteins were performed as described earlier.

To further assess the association of the Ras proteins with the mbranes of Rce1-/- and Rce1+/+ embryonic fibroblasts, we prepared an enhanced green fluorescent protein-Ki-Ras fusion protein in which the last 18 amino acids corresponded to the mouse Ki-Ras sequence. Complementary oligonucleotides corresponding to the last 18 amino acids of mouse Ki-Ras (5'-AATTCTGGTAAAAAGAAGAAAAAGAAGTCAAAGACAAAGTGTGTAATTATGTAGA-3' and 5'-GATCTCTACATAATTACACACTTTGTCTTTGACTTCTTTTTCTTCTTTTTACCAG-3') were annealed and ligated into the vector pEGFP-C1 (CLONTECH, Palo Alto, CA), which had been cleaved with EcoRI and BamHI. The plasmids were then sequenced to verify that the sequence of the oligonucleotide was in frame with that of the green fluorescent protein.

Rce1-/- and Rce1+/+ embryonic fibroblasts were transfected with either 2.0 µg of the the parental green fluorescent protein plasmid (EGFP)-Ki-Ras construct or the EGFP with SuperFect Reagent (Qiagen, Valencia, CA), according to the manufacturer's recommendations. Cells were fixed in 4% formalin 24-28 h after transfection, mounted, and viewed by confocal microscopy.

Adoptive Transfer of Hematopoietic Cells-- Rce1+/- mice were intercrossed to produce Rce1-/-, Rce1+/-, and Rce1+/+ embryos. Pregnant females were sacrificed on day 14.5-15.5 post coitus, and the embryos were removed. Fetal livers were transferred to 1 ml of Iscove's modified Dulbecco's medium supplemented with 20% fetal calf serum. Single-cell suspensions were prepared by drawing the livers through progressively smaller needles (a 22-gauge needle × 2 followed by a 25-gauge needle × 2). DNA was extracted from the remaining embryonic tissues, and a polymerase chain reaction-based assay was used to determine Rce1 genotypes before adoptive transfer. A total of 2-10 × 106 mononuclear cells were injected into the tail veins of recipients previously subjected to 1000 centigrays of total body irradiation. This irradiation protocol destroys recipient hematopoiesis and is associated with >95% survival of animals engrafted with wild-type fetal liver cells. Complete blood counts were performed monthly for at least 4 months after adoptive transfer. The genotype of circulating blood cells was assessed by Southern blot analysis.

To assess the growth of colonies derived from colony-forming unit granulocyte-macrophage progenitors, mononuclear cells from fetal livers were plated on the same day that they were harvested in duplicate 35-mm plates (5 × 104 cells/plate) in fully supplemented Iscove's modified Dulbecco's medium (Stem Cell Technologies, Vancouver, Canada) medium containing 0.8% methylcellulose, 30% fetal calf serum, and L-glutamine. The fetal liver cells and recombinant murine granulocyte-macrophage colony-stimulating factor were added directly to the methylcellulose-containing medium, and the solution was mixed thoroughly before plating. The cells were grown at 37 °C in a humidified 5% CO2 incubator. Colonies derived from granulocyte-macrophage colony forming units were counted by indirect microscopy.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tissue Pattern of Rce1 Gene Expression-- To assess the pattern of Rce1 gene expression in mice, an RNA dot blot was probed with a mouse Rce1 cDNA clone. Rce1 expression was observed in all tissues examined but was particularly high in the heart (Fig. 1). Of note, Rce1 expression was detectable in early stage (E7.0) mouse embryos.


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Fig. 1.   Phosphorimager analysis of an RNA dot blot, illustrating relative levels of Rce1 gene expression in different mouse tissues. A 223-base pair Rce1 cDNA probe was hybridized to a multiple tissue mRNA dot blot. The amount of RNA loaded onto each dot was normalized to the mRNA expression levels of eight housekeeping genes.

Generation of Rce1 Knockout Mice-- Southern blots of EcoRI-, BamHI-, or SacI-cleaved mouse genomic DNA probed with the 1.1-kb EST revealed distinct, single bands (EcoRI, 8.0 kb; BamHI, 10 kb; SacI, 4 kb), suggesting that the mouse genome contains only a single copy of the Rce1 gene (not shown). To inactivate the Rce1 gene, we constructed a sequence replacement gene-targeting vector designed to delete most of the coding sequences of the Rce1 gene (Fig. 2A). After electroporation of the vector into mouse ES cells, Southern blot analysis of 196 drug-resistant ES cell clones revealed that eight were correctly targeted. Two of these clones were used to generate high percentage male chimeric mice, which were bred with C57BL/6 females. Each of the chimeras transmitted the targeted Rce1 mutation to their progeny, generating heterozygous Rce1 knockout mice (Rce1+/-). Male and female Rce1+/- mice were healthy and fertile and were intercrossed to generate homozygotes. From 25 litters, we identified 53 wild-type mice (Rce1+/+), 100 Rce1+/- mice, and 4 knockout homozygotes (Rce1-/-), indicating that homozygosity for the Rce1 mutation caused significant embryonic lethality. All four live-born Rce1-/- mice died, two perinatally and two at 10 days of age. The two mice that survived 10 days were severely runted but otherwise appeared normal. A visual inspection of the major organ systems in one of the Rce1-/- mice revealed no obvious cause of death. The phenotypes of Rce1-deficient mice derived from the two different targeted ES cell clones were identical.


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Fig. 2.   Generation of Rce1-deficient mice. A, a sequence replacement strategy to knock out the Rce1 gene. The assessment of gene-targeting events in ES cells and mouse genotyping were performed by Southern blot analysis, using the indicated probe located 3' to sequences in the gene-targeting vector. B, Southern blot identification of Rce1+/+, Rce1+/-, and Rce1-/- embryos with XbaI-cleaved mouse genomic DNA and the 3'-flanking probe. Also shown is a Southern blot of XbaI-cleaved mouse genomic DNA, using a "deletion" probe spanning the Eco47III-EcoRV fragment of the Rce1 gene that was deleted by the gene-targeting event.

Fig. 2B illustrates the Southern blot identification of Rce1+/- and Rce1-/- mice and also shows a Southern blot with a probe that spanned the segment of the Rce1 gene that was deleted by the knockout mutation. As expected, that probe did not hybridize to any DNA fragment in the Rce1-/- mice.

Rce1 Deficiency Is Lethal in Mice-- To determine the timing of the demise of Rce1-/- embryos, we established 26 timed pregnancies from intercrosses of Rce1+/- mice. Prior to E15.5, Rce1-/- and Rce1+/+ embryos were present in the expected numbers (~25% of the total); however, the percentage of Rce1-/- embryos declined sharply after E15.5 (Fig. 3). Gross inspection of the viable Rce1-/-, Rce1+/+, and Rce1+/- embryos after E15.5 revealed no consistent differences in morphology, organogenesis, stage of development, color, or size. Histological examination of 12.5-, 15.5-, 16.5-, 17.5-, and 18.5-day wild-type (n = 10) and knockout (n = 10) embryos revealed no apparent defect in the development of major organ systems, including the brain, intestinal tract, heart, lungs, kidneys, and placenta. The hearts and thoracic cavities of representative Rce1-/- and Rce1+/+ embryos are shown in Fig. 4 (A-D). High power micrographs of embryonic livers revealed abundant hepatocytes and hematopoietic cells in both Rce1-/- and Rce1+/+ embryos (Fig. 4, E and F).


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Fig. 3.   Percentages of Rce1+/+ and Rce1-/- embryos in litters from intercrosses of Rce1+/- mice at different times during gestation.


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Fig. 4.   Hematoxylin/eosin-stained sections of tissues from Rce1-/- and Rce1+/+ embryos. A, saggital section of the left lateral thoracic cavity from an E15.5 Rce1-/- embryo. B, saggital section of the left lateral thoracic cavity from an E15.5 Rce1+/+ embryo. C, saggital section of the mid-thoracic cavity from an E15.5 Rce1-/- embryo. D, saggital section of the mid-thoracic cavity from an E15.5 Rce1+/+ embryo. E, liver from an E15.5 Rce1-/- embryo. F, liver from an E15.5 Rce1+/+ embryo.

Because the Rce1-/- embryos displayed no overt defect in organogenesis and survived until late in gestation, we suspected that it would be possible to culture embryonic fibroblasts from Rce1-/- embryos. This suspicion was confirmed; the yield and morphology of primary embryonic fibroblasts from E11.5 Rce1-/- and Rce1+/+ embryos were identical. Also, the growth rate of Rce1-/- and Rce1+/+ fibroblasts were indistinguishable when grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum for 26 passages according to a 3T6 protocol (not shown).

The mature erythrocytes in the cardiac chambers of Rce1-/- embryos did not suggest a severe defect in hematopoiesis (Fig. 4, A and C). However, because deficiencies in Ras proteins have been associated with reduced hematopoiesis (35), we further assessed hematopoiesis in the setting of Rce1 deficiency by transplanting Rce1-/- fetal liver hematopoietic stem cells into lethally irradiated mice. There were no gross differences in the appearance of fetal livers of Rce1-/-, Rce1+/-, and Rce1+/+ embryos at days 14.5-15.5 post coitus. However, the number of nucleated cells isolated from Rce1-/- livers was reduced by 37% (range, 12-68% in seven experiments) compared with those from Rce1+/- and Rce1+/+ embryos. Although the absolute number of nucleated cells was less in Rce1-/- embryos, there were no obvious functional deficits in those cells. The dose-response relationship for colony-forming unit granulocyte-macrophage progenitor colony formation from fetal liver cells was similar over a range of granulocyte-macrophage colony-stimulating factor concentrations in all three Rce1 genotypes. Cultures of Rce1-/- fetal liver cells stimulated with saturating amounts of growth factor yielded normal-to-increased numbers of colony-forming unit granulocyte-macrophage colonies, a subset of which showed a distinctive morphology characterized tight clumping of the cells. Rce1-/- fetal liver cells fully reconstituted hematopoiesis in all 20 irradiated recipients. Peripheral blood cell counts (hemoglobin, platelets, lymphocytes, neutrophils, and monocytes) were normal at 1 month after adoptive transfer and were identical in recipients that were injected with Rce1-/-, Rce1+/-, or Rce1+/+ cells (not shown). Blood cell counts remained normal for more than 4 months after adoptive transfer, and Southern blotting of genomic DNA from peripheral blood leukocytes confirmed that Rce1-/- fetal liver cells durably restored hematopoiesis in irradiated recipients (not shown).

Rce1 Deficiency Shifts the Electrophoretic Mobility of Ras Proteins-- To determine whether the mouse Rce1 gene participates in the post-translational processing of Ras proteins, we compared the electrophoretic mobility of the Ras proteins from Rce1-/-, Rce1+/+, and Rce1+/- embryo lysates on SDS-polyacrylamide gels. If the Rce1 gene product were involved in the endoproteolytic processing of Ras, we reasoned that the electrophoretic mobility of the unprocessed Ras proteins in Rce1-/- embryo lysates would likely be retarded. Indeed, this proved to be the case (Fig. 5, A and B). Furthermore, Ras proteins with a normal electrophoretic mobility were undetectable in the Rce1-/- lysates, even after prolonged exposures of the blots. These results strongly suggest that the Rce1 gene product is essential for Ras endoproteolytic processing (at least in E14.5 mouse embryos). Interestingly, the absence of proteolytic processing appeared to have no effect on the levels of Ras proteins in the cells. Also, the electrophoretic mobility of the Ras proteins was identical in Rce1+/+ and Rce1+/- embryo lysates, suggesting that heterozygosity for the Rce1 knockout mutation does not affect Ras processing. To confirm that Ras proteins from Rce1+/+ and Rce1-/- cells truly had different electrophoretic mobilities, we mixed equal amounts of lysates from Rce1-/- and Rce1+/+ embryos and then performed a Western blot for Ras. As expected, two distinct Ras bands were observed in the mixture (Fig. 5C). We also analyzed the electrophoretic mobility of Ras proteins that had been immunoprecipitated from E14.5 embryos and cell lysates with a pan-Ras antibody. Once again, homozygosity for the Rce1 knockout allele resulted in a retarded electrophoretic mobility of the Ras proteins (Fig. 5D).


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Fig. 5.   Abnormal electrophoretic mobility of Ras proteins in lysates from Rce1-/- embryos and primary embryonic fibroblasts. A, a Western blot of lysates of Rce1+/+, Rce1+/-, and Rce1-/- embryos. Lysates were size-fractionated by SDS-polyacrylamide gel electrophoresis, and a Western blot was performed with a pan-Ras-specific monoclonal antibody, Ab-4. B, a Western blot of lysates of Rce1+/+, Rce1+/-, and Rce1-/- embryos with a Ki-Ras-specific monoclonal antibody (Ab-1). C, a Western blot of an equal mixture of Rce1+/+ and Rce1-/- embryo lysates with antibody Ab-4, demonstrating the expected doublet band. D, abnormal electrophoretic mobility of Ras proteins immunoprecipitated from lysates of Rce1-/- embryos and fibroblasts. Ras proteins from Rce1+/+ and Rce1-/- embryos and cultured fibroblasts were immunoprecipitated with a pan-Ras specific antibody; the immunoprecipitate was then analyzed on a Western blot of an SDS-polyacrylamide gel with a pan-Ras-specific antibody.

Absence of CAAX Endoprotease Activity in Rce1-/- Fibroblasts-- The abnormal electrophoretic mobility of the Ras proteins strongly suggested that the Rce1 gene product was responsible for Ras endoproteolytic processing. If so, carboxymethylation of Ras proteins from Rce1-/- cells would be defective because methylation requires prior removal of the three C-terminal amino acid residues. To test that prediction, we metabolically labeled Rce1+/+ and Rce1-/- fibroblasts with L-[methyl-3H]methionine and [35S]cysteine, immunoprecipitated the Ras proteins, and assessed the methylation status of the Ras proteins after fractionating the immunoprecipitates on an SDS-polyacrylamide gel. The amount of [3H]methanol released from the gel slice containing 20-23-kDa proteins by base hydrolysis (which reflects the extent of carboxymethylation of the C-terminal prenyl cysteine) was substantially greater in the Rce1+/+ immunoprecipitates than in the Rce1-/- immunoprecipitates (1013 versus 239 dpm); analysis of 35S incorporation levels showed comparable amounts of Ras protein in the immunoprecipitates from Rce1-/- cells and Rce1+/+ cells (2380 arbitrary units versus 2150 arbitrary units, respectively). In two subsequent experiments, we also observed more base-releasable [3H]methanol from Ras proteins from Rce1+/+ cells than in those from Rce1-/- cells (1557 versus 492 dpm in one experiment, and 349 versus 45 dpm in the other). These data suggested a defect in the carboxymethylation (and thus proteolysis) of Ras proteins in the Rce1-/- fibroblasts.

To directly assess the endoproteolysis of Ras proteins by Rce1+/+ and Rce1-/- fibroblasts, we used an in vitro assay with prenylated recombinant proteins. Initially, we examined the ability of membrane and cytosolic fractions from Rce1+/+ and Rce1-/- fibroblasts to proteolytically process farnesylated Ki-Ras. No proteolysis of the prenylated protein by the cytosolic fraction of Rce1+/+ cells was observed, but the membrane fraction contained robust proteolytic processing activity (Fig. 6A). In contrast, no detectable processing of the farnesylated protein was observed with either the membrane or cytosolic fractions from Rce1-/- fibroblasts. In membranes from Rce1+/+ fibroblasts, prenyl protein endoprotease activity increased linearly with increasing membrane protein concentration, whereas in membranes from Rce1-/- fibroblasts, activity was negligible at all concentrations tested (Fig. 6B). Farnesyl-Ha-Ras, farnesyl-N-Ras, the farnesyl-Ggamma 1 subunit of transducin, geranylgeranyl-Ki-Ras, and geranylgeranyl-Rap1B were each found to be substrates for the protease activity in the membranes from Rce1+/+ fibroblasts, but processing of all these prenylated proteins by the membranes of Rce1-/- fibroblasts was either absent or near the detection limit for this assay (Fig. 6C). The apparent differences in the extent of processing of the different prenylated proteins should not be interpreted as indicating any major differences in their abilities to be recognized by the protease, because, except for Ki-Ras, none of the proteins was purified after in vitro prenylation, and hence their concentrations in the assay varied.


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Fig. 6.   Direct assays of the ability of Rce1-/- fibroblasts to endoproteolytically process prenylated CAAX proteins. A, an assay of the ability of cytosolic and membrane fractions from Rce1-/- fibroblasts to endoproteolytically process farnesyl-Ki-Ras. Membrane and cytosolic fractions (80 µg) from Rce1+/+ and Rce1-/- fibroblasts were incubated for 30 min at 37 °C with farnesyl-Ki-Ras (2 µM) in 100 mM Hepes, pH 7.5, and 5 mM MgCl2. The processed farnesyl-Ki-Ras was then methylated with 5.0 µM [3H]S-adenosylmethionine (1.5 Ci/mmol) and 20 µg of membranes from Sf9 insect cells expressing high concentrations of the yeast prenyl protein carboxymethyltransferase STE14. [3H]Methyl-prenylated proteins were collected on filters, and methylation was quantified by scintillation counting. Proteolysis is described as the number of pmol [3H]methyl groups transferred to the prenylated protein. Each data point was corrected by subtracting levels of [3H]methyl groups trapped on filters in the absence of the recombinant prenylated protein. These experiments were performed twice, with each data point representing duplicate assays. B, concentration dependence of prenyl protein protease activity in the membrane fractions from Rce1+/+ and Rce1-/- fibroblasts. In these experiments, 0-80 µg of fibroblast membranes was analyzed with 2.0 µM farnesyl-Ki-Ras as the prenyl protein substrate. C, endoprotease activity in membranes from Rce1+/+ and Rce1-/- fibroblasts against a panel of prenylated proteins. The prenyl group attached to proteins is indicated by "f-" for farnesyl or by "gg-" for geranylgeranyl. The -AAX sequence for each protein is indicated in italics. The concentrations of farnesyl-Ki-Ras and geranylgeranyl-Ki-Ras (determined by a protein assay) were 1.0 µM. The approximate concentration (determined by exhaustive proteolysis and methylation of each substrate) of geranylgeranyl-Rap1B was 0.2 µM, and the approximate concentrations of farnesyl-Ha-Ras, farnesyl-N-Ras, and farnesyl-Ggamma 1 were 1 µM.

Rce1 Deficiency Results in a Mislocalization of Ras Proteins-- To test whether the absence of Ras proteolytic processing and methylation altered the intracellular localization of Ras, we compared the relative levels of Ras proteins in the membrane and cytosolic fractions of Rce1-/- and Rce1+/+ embryonic fibroblasts with immunoblotting techniques. In spontaneously immortalized fibroblasts, virtually all of the Ras in Rce1+/+ cells was located in the membrane fractions (Fig. 7). In contrast, approximately 50% of the Ras within Rce1-/- cells was in the cytosolic fraction (Fig. 7). This experiment was repeated with early passage primary embryonic fibroblasts. Once again, there was an increased percentage of the Ras proteins in the cytosolic fractions of Rce1-/- cells (not shown).


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Fig. 7.   Intracellular localization of Ras proteins in immortalized Rce1+/+ and Rce1-/- fibroblasts. Cells were fractionated into cytosolic (S100) and membrane (P100) fractions by ultracentrifugation. Ras proteins were immunoprecipitated from the cytosolic and membrane fractions, as well as from the total cellular lysates, and then analyzed on a Western blot of an SDS-polyacrylamide gel with the pan-Ras-specific antibody Ab-4.

To further address the issue of Ras localization within cells, both Rce1-/- and Rce1+/+ cells were transfected with a green fluorescent protein-Ras fusion protein in which the C-terminal 18 amino acids were identical to mouse Ki-Ras (including the CAAX sequence). The fluorescence was highly localized to the plasma membrane in Rce1+/+ cells (Fig. 8, A-C) but was largely cytoplasmic in the Rce1-/- cells (Fig. 8, D-F). As a control, we transfected EGFP into both Rce1+/+ and Rce1-/- cells. As expected, the green fluorescent protein was distributed uniformly in the cytoplasm of both cell types (Fig. 8, G-I).


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Fig. 8.   Confocal microscopic images of Rce1+/+ fibroblasts (A-C) and Rce1-/- fibroblasts (D-F) transfected with a plasmid encoding an EGFP-Ki-Ras fusion protein. G, Rce1+/+ fibroblasts transfected with the parental EGFP plasmid; H and I, Rce1-/- cytosolic fibroblasts transfected with the EGFP plasmid.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our studies show that the Rce1 gene product is essential for the endoproteolytic processing of all of the mammalian Ras proteins. Three lines of evidence support this statement. First, Ras proteins with a normal electrophoretic mobility (indicating normal processing) were undetectable in Rce1-/- embryos or fibroblast lysates. Second, metabolic labeling experiments revealed that the Ras proteins from Rce1-/- fibroblasts could not be carboxymethylated, a processing step that requires prior proteolysis. Third and most important, the endoproteolytic processing of prenylated recombinant Ras proteins by membranes from Rce1--/- fibroblasts was undetectable.

Two findings from the in vitro assays of endoproteolytic processing deserve comment. First, the membranes from Rce1-deficient fibroblasts were defective in processing both farnesylated and geranylgeranylated proteins, indicating that this protease recognizes both classes of prenylated CAAX proteins. Second, neither geranylgeranylated Rap1B nor farnesylated Ggamma 1 was processed by Rce1-/- fibroblast membranes, strongly suggesting that the Rce1 gene product plays a broad role in processing prenylated CAAX proteins.

The principal finding our study, that Rce1 is critical for the processing of a group of prenylated proteins, is entirely consistent with the conclusions of a companion study by Otto and co-workers (33). They cloned the human RCE1 cDNA and expressed it at high levels in Sf9 insect cells by infection with a recombinant baculovirus. The membrane preparations derived from the transfected Sf9 cells exhibited a high level of prenyl protease activity and were capable of processing both farnesylated and geranylgeranylated proteins, including the Ras proteins, Rap1B, and the heterotrimeric G protein Ggamma 1 subunit.

Whether the product of the Rce1 gene processes all of the mammalian CAAX proteins is not known. In yeast, Afc1p cleaves the -AAX from the precursor of the a-mating factor. Recently, Tam and co-workers (20) reported the cDNA sequence of the human homologue of yeast AFC1 and demonstrated that the human Afc1 protein has the capacity to process yeast a-mating factor and to complement the mating defect of yeast lacking both Afc1p and Rce1p. However, a human homologue for yeast a-mating factor has not been identified, and no substrates for the human AFC1 gene product are known. Defining the unique and/or overlapping biochemical specificities for the mammalian Afc1 and Rce1 proteins in processing mammalian CAAX proteins will be of great interest.

A deficiency in Rce1 gene expression in mice is incompatible with survival. The vast majority of Rce1-/- embryos die late in gestation, and the few mice that are born alive are severely runted and die shortly after birth. It is not clear why the Rce1-/- embryos do not survive, in view of the fact that all of the major organ systems developed and were free of obvious pathology. Mutations that cause death late in mouse embryonic development often involve disorders of the circulation (e.g. defective hematopoiesis or a defective heart or placenta) (36). However, the placenta of Rce1-deficient embryos appeared normal; the liver contained large numbers of hematopoietic precursor cells; the heart and great vessels were filled with mature erythrocytes; the heart was normal in size; and the cardiac myocytes appeared histologically normal. In addition, the rescue of lethally irradiated mice by Rce1-/- hematopoietic stem cells makes it unlikely that the Rce1-deficient mice die from hematopoietic failure. Although the precise cause of death of these animals remains unclear, we suggest that the failure to process some population of prenylated CAAX proteins might render certain cellular metabolic functions inefficient, perhaps in a tissue-specific manner, leading to death without pathological changes that are obvious by light microscopy.

In yeast, a deficiency in Rce1p interferes with Ras2p processing, causes a mislocalization of a Ras2p-green fluorescent protein fusion to the cytoplasm, and blocks the heat sensitivity caused by a mutationally activated Ras protein. However, the deficiency in Rce1p does not affect the growth of yeast under normal conditions (19). An absence of Rce1p in yeast likely modulates Ras function rather than blocking it completely. Of note, the rce1Delta yeast grew normally, whereas the deletion of both yeast Ras genes (RAS1 and RAS2) is lethal (37). Also, the mislocalization of the Ras2p fusion in rce1Delta yeast did not appear to be absolute; a fraction of the Ras2p fusion reached the plasma membrane (19). Our studies of Rce1-deficient mice suggest intriguing similarities to the situation in rce1Delta yeast. As in rce1Delta yeast, the processing of Ras proteins in Rce1-deficient embryos and fibroblasts was completely blocked, and Ras proteins were mislocalized within cells. Also, the elimination of Rce1 enzymatic activity had no obvious effects on cellular growth. Indeed, the growth of cultured Rce1-/- fibroblasts was quite normal, and the growth of Rce1-/- mouse embryos appeared to be normal through E15. The relatively normal development of the Rce1-/- mouse embryos indicated that Ras function was probably not completely blocked, because a knockout of the Ki-Ras gene alone causes death between E13 and E15 (35, 38), and a deficiency of both Ki-Ras and N-Ras caused defective hematopoiesis and embryonic death around E12 (35). Thus, as in yeast, we suggest that Rce1 deficiency in mice modulates rather than blocks Ras function. The precise extent to which Rce1 deficiency interferes with Ras function in mammalian cells is not clear and requires further study; however, the experimental tools developed in this study, including the Rce1-deficient fibroblasts and the adoptive bone marrow transfer model, should be helpful in addressing that issue. For example, competitive repopulation assays will allow us to ascertain if inactivation of Rce1 confers a proliferative defect in specific lineages of hematopoietic cells.

Mutations that lead to constitutively active Ras proteins cause increased cell growth and division and are involved in the pathogenesis of many human cancers (39). The frequent involvement of mutationally activated Ras proteins in human cancers, combined with the requirement for C-terminal post-translational processing for Ras function, has prompted efforts to interfere with the processing of the Ras proteins and thereby impede the growth of cancers. Most of this effort has focused on developing drugs to block protein farnesyltransferase (FTase) and thereby interfere with the farnesylation of Ras. Potent inhibitors of FTase have been developed and have proved to be effective, both in cell culture systems and in animal models, in retarding the growth of a wide variety of cancers. However, some Ras proteins, including Ki-Ras, the one most commonly activated by somatic mutation in human cancers, are geranylgeranylated by protein geranylgeranyltransferase I in the setting of FTase inhibitors (40). This alternate pathway for prenylation might limit the efficacy of FTase inhibitors against many cancers. The development of Rce1 inhibitor drugs could provide another approach for reducing the activity of Ras and would be expected to interfere with the processing of the Ras proteins regardless of whether they were farnesylated or geranylgeranylated. Of course, it remains to be determined whether any particular Rce1 inhibitor drug would be clinically effective, but the normal blood counts in mice rescued with Rce1-deficient hematopoietic cells suggest that Rce1 inhibitor drugs might be free of one side effect of many anticancer drugs, myelosuppression. Interestingly, FTase inhibitors have been effective against cancers that lack a mutationally activated Ras, suggesting that inhibiting the activity of other farnesylated proteins, aside from Ras proteins, may be important in inhibiting tumor growth (41). Those observations are intriguing and suggest that inhibitors of Rce1 might be active in blocking tumor growth (either alone or in combination with FTase inhibitors) by acting against a range of different CAAX proteins.

    ACKNOWLEDGEMENTS

We thank G. Howard and S. Ordway for comments on the manuscript, S. Clarke for advice regarding the carboxymethylation experiments, and D. Sanan for assistance with confocal microscopy.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL-41633, HL-47660, and AG-15451 (to S. G. Y.) and CA-72614 (to K. S.), by American Cancer Society Grant BE 117 (to P. J. C.), and by Acacia Biosciences.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel Supported by a Postdoctoral Fellowship Award for Physicians from the Howard Hughes Medical Institute. To whom correspondence should be addressed: Gladstone Inst. of Cardiovascular Disease, P. O. Box 419100, San Francisco, CA 94141-9100. Tel.: 415-826-7500; Fax: 415-285-5632; E-mail: ekim{at}gladstone.ucsf.edu.

Dagger Dagger Recipient of a Leukemia Society of America postdoctoral fellowship.

    ABBREVIATIONS

The abbreviations used are: EST, expressed sequence tag; ES, embryonic stem; kb, kilobase(s); En, embryonic day n; PBS, phosphate-buffered saline; FTase, farnesyltransferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Clarke, S., Vogel, J. P., Deschenes, R. J., and Stock, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4643-4647[Abstract]
  2. Zhang, F. L., and Casey, P. J. (1996) Annu. Rev. Biochem. 65, 241-269[CrossRef][Medline] [Order article via Infotrieve]
  3. Schafer, W. R., and Rine, J. (1992) Annu. Rev. Genet. 30, 209-237
  4. Glomset, J. A., and Farnsworth, C. C. (1994) Annu. Rev. Cell Biol. 10, 181-205[CrossRef]
  5. Casey, P. J., and Seabra, M. C. (1996) J. Biol. Chem. 271, 5289-5292[Free Full Text]
  6. Finegold, A. A., Johnson, D. I., Farnsworth, C. C., Gelb, M. H., Judd, S. R., Glomset, J. A., and Tamanoi, F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4448-4452[Abstract]
  7. Moores, S. L., Schaber, M. D., Mosser, S. D., Rands, E., O'Hara, M. B., Garsky, V. M., Marshall, M. S., Pompliano, D. L., and Gibbs, J. B. (1991) J. Biol. Chem. 266, 14603-14610[Abstract/Free Full Text]
  8. Schmidt, W. K., Tam, A., Fujimura-Kamada, K., and Michaelis, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11175-11180[Abstract/Free Full Text]
  9. Clarke, S. (1993) Curr. Opin. Cell Biol. 5, 977-983[Medline] [Order article via Infotrieve]
  10. Clarke, S. (1992) Annu. Rev. Biochem. 61, 355-386[CrossRef][Medline] [Order article via Infotrieve]
  11. Hancock, J. F., Magee, A. I., Childs, J. E., and Marshall, C. J. (1989) Cell 57, 1167-1177[Medline] [Order article via Infotrieve]
  12. Brown, M. S., and Goldstein, J. L. (1993) Nature 366, 14-15[CrossRef][Medline] [Order article via Infotrieve]
  13. Dai, Q., Choy, E., Chiu, V., Romano, J., Slivka, S. R., Steitz, S. A., Michaelis, S., and Philips, M. R. (1998) J. Biol. Chem. 273, 15030-15034[Abstract/Free Full Text]
  14. Hrycyna, C. A., and Clarke, S. (1992) J. Biol. Chem. 267, 10457-10464[Abstract/Free Full Text]
  15. Rando, R. R. (1996) Biochim. Biophys. Acta 1300, 5-16[Medline] [Order article via Infotrieve]
  16. Gilbert, B. A., Ma, Y.-T., and Rando, R. R. (1995) Methods Enzymol. 250, 206-215[Medline] [Order article via Infotrieve]
  17. Ashby, M. N., King, D. S., and Rine, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4613-4617[Abstract]
  18. Ma, Y.-T., and Rando, R. R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6275-6279[Abstract]
  19. Boyartchuk, V. L., Ashby, M. N., and Rine, J. (1997) Science 275, 1796-1800[Abstract/Free Full Text]
  20. Tam, A., Nouvet, F. J., Fujimura-Kamada, K., Slunt, H., Sisodia, S. S., and Michaelis, S. (1998) J. Cell Biol. 142, 635-649[Abstract/Free Full Text]
  21. Fujimura-Kamada, K., Nouvet, F. J., and Michaelis, S. (1997) J. Cell Biol. 136, 271-285[Abstract/Free Full Text]
  22. Boyartchuk, V. L., and Rine, J. (1998) Genetics 150, 95-101[Abstract/Free Full Text]
  23. Jackson, J. H., Cochrane, C. G., Bourne, J. R., Solski, P. A., Buss, J. E., and Der, C. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3042-3046[Abstract]
  24. Silvius, J. R., and l'Heureux,, F. (1994) Biochemistry 33, 3014-3022[Medline] [Order article via Infotrieve]
  25. Hancock, J. F., Cadwallader, K., and Marshall, C. J. (1991) EMBO J. 10, 641-646[Abstract]
  26. Hrycyna, C. A., and Clarke, S. (1993) Pharmacol. Ther. 59, 281-300[CrossRef][Medline] [Order article via Infotrieve]
  27. Kato, K., Cox, A. D., Hisaka, M. M., Graham, S. M., Buss, J. E., and Der, C. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6403-6407[Abstract]
  28. Gelb, M. H. (1997) Science 275, 1750-1751[Free Full Text]
  29. Hanks, M., Wurst, W., Anson-Cartwright, L., Auerbach, A. B., and Joyner, A. L. (1995) Science 269, 679-682[Medline] [Order article via Infotrieve]
  30. McMahon, A. P., and Bradley, A. (1990) Cell 62, 1073-1085[Medline] [Order article via Infotrieve]
  31. Farese, R. V., Jr., Cases, S., Ruland, S. L., Kayden, H. J., Wong, J. S., Young, S. G., and Hamilton, R. L. (1996) J. Lipid Res. 37, 347-360[Abstract]
  32. Todaro, G. J., and Green, H. (1963) J. Cell Biol. 17, 299-313[Abstract/Free Full Text]
  33. Otto, J. C., Kim, E., Young, S. G., and Casey, P. J. (1999) J. Biol. Chem. 274, 8379-8382[Abstract/Free Full Text]
  34. Cox, A. D., Solski, P. A., Jordan, J. D., and Der, C. J. (1995) Methods Enzymol. 255, 195-220[Medline] [Order article via Infotrieve]
  35. Johnson, L., Greenbaum, D., Cichowski, K., Mercer, K., Murphy, E., Schmitt, E., Bronson, R. T., Umanoff, H., Edelmann, W., Kucherlapati, R., and Jacks, T. (1997) Genes Dev. 11, 2468-2481[Abstract/Free Full Text]
  36. Copp, A. J. (1995) Trends Genet. 11, 87-93[CrossRef][Medline] [Order article via Infotrieve]
  37. Kataoka, T., Powers, S., McGill, C., Fasano, O., Strathern, J., Broach, J., and Wigler, M. (1984) Cell 37, 437-445[Medline] [Order article via Infotrieve]
  38. Koera, K., Nakamura, K., Nakao, K., Miyoshi, J., Toyoshima, K., Hatta, T., Otani, H., Aiba, A., and Katsuki, M. (1997) Oncogene 15, 1151-1159[CrossRef][Medline] [Order article via Infotrieve]
  39. Barbacid, M. (1987) Annu. Rev. Biochem. 56, 779-827[CrossRef][Medline] [Order article via Infotrieve]
  40. Whyte, D. B., Kirschmeier, P., Hockenberry, T. N., Nunez-Oliva, I., James, L., Catino, J. J., Bishop, W. R., and Pai, J.-K. (1997) J. Biol. Chem. 272, 14459-14464[Abstract/Free Full Text]
  41. Gibbs, J. B., Graham, S. L., Hartman, G. D., Koblan, K. S., Kohl, N. E., Omer, C. A., and Oliff, A. (1997) Curr. Opin. Chem. Biol. 1, 197-203[CrossRef][Medline] [Order article via Infotrieve]


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