CHARACTERIZATION OF HA-RAS, N-RAS, KI-RAS4A, AND KI-RAS4B AS IN-VITROSUBSTRATES FOR FARNESYL-PROTEIN TRANSFERASE AND GERANYLGERANYL PROTEIN TRANSFERASE TYPE-I

Ras proteins are small GTP-binding proteins which are critical for cel l 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, 62 21-6226), 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 e quimolar 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 high est affinity substrate for GGPT-1 and is similar in affinity to a stan dard GGPT-1 substrate terminating in leucine, However, the catalytic e fficiencies of these geranylgeranylation reactions are between 15- and 140-fold lower than the corresponding farnesylation reactions, largel y reflecting differences in affinity. Carboxyl-terminal peptides accou nt for many of the properties of the Ras proteins, One interesting exc eption is that, unlike the full-length N-Ras protein, a carboxyl-termi nal N-Ras peptide is not a GGPT-1 substrate, raising the possibility t hat upstream sequences in this protein may play a role in its recognit ion by GGPT-1. Studies with various carboxyl-terminal peptides from Ki -Ras4B suggest that both the carboxyl-terminal methionine and the upst ream polylysine region are important determinants for geranylgeranylat ion. Furthermore, it was found that full-length Ki-Ras4B, but not othe r Ras isoforms, can be geranylgeranylated in vitro by FPT. These findi ngs suggest that the different distribution of Ras isoforms and the ab ility of cells to alternatively process these proteins mag explain in part the resistance of some cell lines to FPT inhibitors.