Characterization of Ha-Ras, N-Ras, Ki-Ras4A, and Ki-Ras4B as in Vitro Substrates for Farnesyl Protein Transferase and Geranylgeranyl Protein Transferase Type I

Abstract

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...

Growth inhibition of astrocytoma cells by farnesyl transferase inhibitors is mediated by a combination of anti-proliferative, pro-apoptotic and anti-angiogenic effects

“…The IC 50 doses for our cell lines are however clearly higher than has been demonstrated for H-Rastransformed ®broblasts (Kohl et al, 1993. FTIs preferentially inhibit the proliferation of H-Ras transformed cells, with K-Ras transformed cells either being resistant to these agents or being inhibited at higher drug doses (James et al, 1996;Lerner et al, 1995b;Manne et al, 1995;Nagasu et al, 1995;Zhang et al, 1997). This relative resistance of K-Rastransformed cell lines to FTIs is due to the observation that K-Ras and (to a lesser extent) NRas, but not H-Ras, can be post-translationally modi®ed by geranylgeranyltransferase I (GGTase I) (Lerner et al, 1995b;Whyte et al, 1997), a related enzyme which transfers a 20-carbon prenyl group from geranylgeranyl pyrophosphate to cysteine residues in proteins.…”

The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

“…19). The cysteine in the CAAX box is recognized by farnesyltransferases that catalyze the addition of a farnesyl isoprenoid, [20] which permits a transient association to the ER. For stable membrane association Ras requires a second signal: H-Ras, N-Ras, and K-Ras4A are further modified by one or two palmitates.…”

Bcl-2 differentially targets K-, N-, and H-Ras to mitochondria in IL-2 supplemented or deprived cells: Implications in prevention of apoptosis

“…In contrast, K-Ras proteins are not palmitoylated, but possess a polybasic domain that can be reversibly phosphorylated (Glomset andFarnsworth, 1994, Egan et al, 1993). Ras homologues also di er in their a nity for FPT in vitro and in their sensitivity to FPT inhibition, since K-and N-Ras can be alternatively geranylgeranylated in cells treated with FPT inhibitors (Whyte et al, 1997;Lerner et al, 1997;Zhang et al, 1997).…”