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 (Km = 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 carboxyl-terminal 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.
Highlights
Ras proteins are small GTP-binding proteins that play critical roles in cell signaling, differentiation, and proliferation [1]
In the studies reported here, we examined for the first time all four full-length Ras isoforms as substrates for the prenyltransferases, farnesyl protein transferase (FPT) and geranylgeranyl protein transferase-1 (GGPT-1)
Materials—Recombinant human FPT was expressed and purified (Ͼ95% pure) from the baculovirus/Sf9 cell expression system according to procedures described previously [31]. [1-3H]Farnesyl diphosphate (22.5 Ci/mmol) and [1-3H]geranylgeranyl diphosphate (19.5 Ci/mmol) were obtained from DuPont NEN
Summary
Materials—Recombinant human FPT was expressed and purified (Ͼ95% pure) from the baculovirus/Sf9 cell expression system according to procedures described previously [31]. [1-3H]Farnesyl diphosphate (22.5 Ci/mmol) and [1-3H]geranylgeranyl diphosphate (19.5 Ci/mmol) were obtained from DuPont NEN. To produce recombinant GGPT-1, a cDNA clone for the human FPT ␣ subunit was obtained from ATCC (ATCC63225) This cDNA was excised with BamHI and PpuMI, and the resulting fragment was ligated into the vector p2-BAC (Invitrogen) to generate the construct pA2BN. For GGPT-1 production, log phase Sf9 cells (2 ϫ 106/ml) were infected at a multiplicity of We show the carboxyl-terminal sequences from the 4 Ras isoforms and various peptide substrates derived from Ki-Ras4B and used in the experiments reported here. The standard assay mixture contained 50 mM Tris1⁄7HCl (pH 7.5), 1 mM dithiothreitol, 20 mM KCl, 50 –2000 nM biotinylated peptide, 0.5 mM Zwittergent 3-14, 200 nM [3H]FPP (22.5 Ci/mmol) or [3H]GGPP (19.5 Ci/mmol), and 25 ng of purified recombinant FPT or 40 ng of GGPT-1 in a final volume of 50 l.
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