The first step in Ras post-translational modifications that leads to membrane association is farnesylation (29). This lipid modification is catalyzed by farnesyltransferase (FTase), which transfers farnesyl from farnesylpyrophosphate (FPP), a cholesterol biosynthesis intermediate (30), to the cysteine of the carboxyl terminal CAAX tetrapeptide ofRas (C = Cys; A = any amino acid; and X = Met or Ser) (29). Farnesylation is followed by peptidase cleavage ofAAX and carboxymethylation ofthe resulting farnesylated cysteine (22,23). H-, N-, and Ka-Ras proteins are also palmitoylated on cysteines upstream of the farnesylated cysteine and these additional lipid modifications further stabilize the interaction of Ras with the plasma membrane (22,23,26). Kb-Ras on the other hand, is not palmitoylated, but contains apolylysine stretch upstream ofthe farnesylated cysteine that further stabilizes the interaction of Kb-Ras with the plasma membrane (26). Farnesylation is aprerequisite for all subsequent Ras post-translational modifications. Furthermore, farnesylation was shown to be absolutely required for Ras malignant transformation (25,31).
In addition to the Ras proteins, FTase farnesylates several other cellular proteins (29,32,33). The protein substrates of FTase all share a common feature: a CAAX at their carboxyl terminal, where X is most often amethionine, serine, cysteine, alanine, or gluta-mine (29,32,33). Proteins terminating with CAAX boxes, where X is leucine or isoleucine, are modified with the 20-carbon cholesterol biosynthesis intermediate geranylgeranyl pyrophosphate (GGPP) (27,34,35). The enzyme responsible for this post-translational modification is geranylgeranyltransferase I (GGTase I). FTase and GGTase I are a/p heterodimeric zinc metalloproteins that share the a-subunit (36,37). The p-subunit of FTase has been shown to bind the protein substrate, as well as the prenyl substrate FPP. Among the substrates for FTase are Ras proteins, lamin B and transducin (27). GGTase I substrates are more numerous and include most y-subunits of heterotrimeric G-proteins and a large number of small G-proteins such as those in the Rho family (27). A member of this family, Rho B, was shown to be geranylgeranylated and farnesylated (38-40). Although H-Ras is exclusively farnesylated, recent in vitro and in vivo data suggested that Kb-Ras andN-Ras become geranylgeranylated when FTase is inhibited (41-45). For more detail about the biochemistry of FTase and GGTase I, please refer to Chapter 2.
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