The association of mutant forms of Ras protein with a variety of human cancers has stimulated intense interest in therapies based on inhibiting oncogenic Ras signaling. Attachment of Ras proteins to the plasma membrane is required for effective Ras signaling and is initiated by the enzyme farnesyl protein transferase. We found that in the presence of potent farnesyl protein transferase inhibitors, Ras proteins in the human colon carcinoma cell line DLD-1 were alternatively prenylated by geranylgeranyl transferase-1. When H-Ras, N-Ras, K-Ras4A, and K-Ras4B were expressed individually in COS cells, H-Ras prenylation and membrane association were found to be uniquely sensitive to farnesyl transferase inhibitors; N-and K-Ras proteins incorporated the geranylgeranyl isoprene group and remained associated with the membrane fraction. The alternative prenylation of N-and K-Ras has significant implications for our understanding of the mechanism of action of farnesyl protein transferase inhibitors as anti-cancer chemotherapeutics.Newly synthesized Ras proteins are partitioned to the cytoplasmic face of the plasma membrane by a series of posttranslational modifications. The first step, catalyzed by the enzyme farnesyl protein transferase, is the addition of the 15-carbon isoprenyl group farnesyl to the sulfhydryl group of cysteine in the Ras carboxyl-terminal CAAX box (where C is cysteine, A is aliphatic, and X is typically Met or Ser) (1-3). Farnesylation is followed by proteolytic removal of the AAX amino acids and methylation of the carboxyl group of the farnesylated cysteine (4). Ras proteins at the plasma membrane cycle between an active GTP-bound state and an inactive GDPbound state. Mutations that stabilize the active GTP-bound state have been identified in over 30% of human tumors, with particularly high incidences in pancreatic (ϳ90%) and colon (ϳ50%) cancers. Four oncogenic Ras proteins have been described, H-Ras, N-Ras, K-Ras4A, and K-Ras4B. The majority of mutations associated with human cancer have been found in the K-Ras gene. The two K-Ras proteins are products of a single alternatively spliced transcript, with K-Ras4B the predominant isoform (Ͼ80%) (5, 6).Ras proteins that have been genetically modified so that they lack the isoprenylated cysteine do not associate with the plasma membrane and cannot transform fibroblasts (7). These genetic experiments provided the basis for the development of farnesyl transferase inhibitors (FTIs) 1 as anti-cancer agents. A number of reports have demonstrated that pharmacological inhibition of farnesyl protein transferase by CAAX analogs reduces anchorage-independent growth of Ras-transformed cells in soft agar (8) and slows growth of Ras-transformed cells in nude mice (9, 10). The FTIs appear relatively non-toxic in that they do not interfere with normal cell proliferation (11). This result was somewhat surprising because Ras function was shown to be necessary for normal growth factor signaling and cell proliferation (12). A mechanism through which cells may proliferate in...

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Ras proteins are small GTP-binding proteins which are critical for cell signaling and proliferation. Four Ras isoforms exist: Ha-Ras, N-Ras, Ki-Ras4A, and Ki-Ras4B. The carboxyl termini of all four isoforms are post-translationally modified by farnesyl protein transferase (FPT). Prenylation is required for oncogenic Ras to transform cells. Recently, it was reported that Ki-Ras4B is also an in vitro substrate for the related enzyme geranylgeranyl protein transferase-1 (GGPT-1) (James, G. L., Goldstein, J. L., and Brown, M. S. (1995) J. Biol. Chem. 270, 6221-6226). In the current studies, we compared the four isoforms of Ras as substrates for FPT and GGPT-1. The affinity of FPT for Ki-Ras4B (K m ‫؍‬ 30 nM) is 10 -20-fold higher than that for the other Ras isoforms. Consistent with this, when the different Ras isoforms are tested at equimolar concentrations, it requires 10 -20-fold higher levels of CAAX-competitive compounds to inhibit Ki-Ras4B farnesylation. Additionally, we found that, as reported for Ki-Ras4B, N-Ras and Ki-Ras4A are also in vitro substrates for GGPT-1. Of the Ras isoforms, N-Ras is the highest affinity substrate for GGPT-1 and is similar in affinity to a standard GGPT-1 substrate terminating in leucine. However, the catalytic efficiencies of these geranylgeranylation reactions are between 15-and 140-fold lower than the corresponding farnesylation reactions, largely reflecting differences in affinity. Carboxyl-terminal peptides account for many of the properties of the Ras proteins. One interesting exception is that, unlike the full-length N-Ras protein, a carboxylterminal N-Ras peptide is not a GGPT-1 substrate, raising the possibility that upstream sequences in this protein may play a role in its recognition by GGPT-1. Studies with various carboxyl-terminal peptides from Ki-Ras4B suggest that both the carboxyl-terminal methionine and the upstream polylysine region are important determinants for geranylgeranylation. Furthermore, it was found that full-length Ki-Ras4B, but not other Ras isoforms, can be geranylgeranylated in vitro by FPT. These findings suggest that the different distribution of Ras isoforms and the ability of cells to alternatively process these proteins may explain in part the resistance of some cell lines to FPT inhibitors.Ras proteins are small GTP-binding proteins that play critical roles in cell signaling, differentiation, and proliferation (1). Ras signaling is regulated by a GDP-GTP cycle. Binding of GTP to Ras is required for its productive interaction with Raf-1 and other downstream effector proteins (2). Ras proteins are activated by nucleotide exchange factors such as SOS-1 which stimulate the exchange of GDP for GTP. The lifetime of activated Ras is limited by its intrinsic GTPase activity, which hydrolyzes GTP to GDP. GTPase-activating proteins, such as p120 Ras-GAP and NF-1, stimulate this activity and thereby facilitate inactivation of Ras proteins (2). Transforming mutations of Ras which decrease the rate of GTP hydrolysis result in its constitutive activation. S...

Oncogenic forms of Ras proteins are associated with a broad range of human cancers including an estimated 90% of all colon cancers (1). Ras proteins undergo a complex series of posttranslational processing events, which have been defined over the past several years (2, 3). The initial post-translational event is the transfer of the 15-carbon isoprene farnesyl from farnesyl pyrophosphate to a Cys residue (Cys 186 in Ha-Ras) in the conserved carboxyl-terminal "CAAX" motif (where "A" is an aliphatic residue) present in all Ras proteins (4, 5). Studies employing site-directed mutagenesis (6, 7) or inhibitors of hydroxymethylglutaryl-CoA reductase (8), the rate-limiting enzyme in isoprenoid biosynthesis, demonstrated that isoprenylation is required for Ras proteins to become membraneassociated and to induce cellular transformation. The farnesyl protein transferase (FPT) 1 that catalyzes this reaction has been purified (9) and cDNA clones for its ␣ and ␤ subunits isolated (10 -12).A number of other cellular proteins are also isoprenylated on a Cys residue near their COOH terminus (13,14). These include other substrates for FPT, such as the nuclear lamins (15). However, the majority of cellular isoprenylated proteins are modified with geranylgeranyl, a 20-carbon isoprene. Two distinct geranylgeranyl protein transferases (GGPT I and II) have been identified (16,17) and cDNA clones for their ␣ and ␤ subunits isolated (18,19). GGPT I and FPT share a common ␣ subunit (18,20).The primary determinant for recognition of protein substrates by the isoprenyl transferases is the substrate's carboxyl-terminal amino acid sequence. Proteins ending in Cys-X-XSer (or Met) are preferred substrates for FPT, while proteins terminating in Cys-X-X-Leu are preferred substrates for GGPT I (21, 22). Substitution of leucine for serine at the COOH terminus of the Ha-Ras CAAX box (Ser 189 3 Leu) makes this protein a substrate for geranylgeranylation (rather than farnesylation) both in vitro and in cells (23). The different substrate specificities of FPT and GGPT-1 are likely mediated by their distinct ␤ subunits. GGPT II utilizes protein substrates terminating in Cys-Cys or Cys-X-Cys (17,24).A number of inhibitors of FPT have been reported over the past several years (25). The design of CAAX peptidomimetics (26 -29) has resulted in potent and selective FPT inhibitors capable of blocking Ras processing in cells. These compounds have shown considerable promise as antitumor agents based on their ability to inhibit cellular transformation induced by oncogenic Ras proteins (26,27) and the growth of Ras-dependent

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