RAS and many other oncogenic proteins undergo a complex series of post-translational modifications that are initiated by the addition of farnesyl or a geranyl-geranyl moiety to C-terminal cysteine(s) of the target protein through a process known as prenylation.
Fujiyama A in 1991 demonstrate that polyisoprenylation and methyl esterification of the cysteine residue in the C-terminal domain of the RAS2 protein are involved in the conversion process from precursor form to intermediate form. The polyisoprenoid moiety attached to the RAS2 protein was
identified as a 15-carbon farnesyl group through two independent experiments: the release of S-farnesylcysteine with carboxypeptidase Y from the RAS2 protein, and the recovery of radioactive farnesol through methyliodide treatment of the RAS2 protein purified. The farnesyl group attached to the RAS2 protein was detected predominantly in the C-terminal peptide 1. The mam4 mutation of Schizosaccharomyces pombe causes mating deficiency in h2 cells but not in h1 cells. h2 cells defective in mam4 do not secrete active mating pheromone M-factor. Xmam4 was shown to have a farnesyl cysteine carboxyl methyltransferase activity in S. pombe cells 2.
Eukaryotic polypeptides synthesized with the C-terminal sequence -Cys-Xaa-Xaa-Xaa (where Xaa is any amino acid) can be posttranslationally modified by proteolytic, lipidation, and methylation reactions. a peptide necessary for mating between haploid a and a cells of the yeast S. cerevisiae. At least three genes have been associated with its posttranslational maturation, which results in a C-terminal S-farnesyl cysteine a-methyl ester residue. The resulting structures,
first found in the peptidyl mating factors from the jelly fungi Tremella mesenterica and Tremella brasiliensis, contain C-terminal cysteine residues that are modified by both an S-isoprenyl group in a thioether linkage and a-methyl esterification. Additional examples include the a mating factor and RAS proteins from Saccharomyces cerevisiae and the mammalian Ras proteins, large G-proteins, cyclic GMP phosphodiesterase and nuclear lamins 3. The mating pheromones of S.pombe, namely, P-factor secreted by h1 cells and M-factor secreted by h2 cells, mediate cell-cell interaction prior to mating. While P-factor is a peptide of 23 amino acids that does not appear to be modified, M-factor is a nonapeptide whose C-terminal cysteine residue is both farnesylated and carboxylmethylated and resembles S. cerevisiae alpha-factor 4.
Mode of Action
A number of proteins, including Ras superfamily GTP-binding proteins, are processed from precursors carrying a CAAX motif at their C termini. C, A, and X in the motif represent, respectively, cysteine, an aliphatic amino acid, and an unspecified amino acid. The precursors undergo three steps of posttranslational modifications at this motif to generate mature proteins. First, the cysteine residue is prenylated, i.e., either farnesylated or geranylgeranylated, and this is followed by removal of the C-terminal three residues, AAX. The new Cterminal residue, prenylated cysteine, is then methylesterified. These modifications are thought to be important to localize the proteins to the membrane. A membrane-associated activity that methylesterifies prenylcysteine has been detected in S. cerevisiae and mammals and a single activity appears to methylesterify both farnesyl cysteine and geranylgeranyl cysteine. Carboxylmethylation is reversible under physiological conditions, raising the interesting possibility that methylation also regulate a biological activity of the target proteins 5,6,7.
RAS proteins usually undergo endoproteolytic processing by the RCE1 protease and then carboxyl methylation by a unique methyltransferase known as isoprenylcysteine carboxyl methyltransferase (ICMT). Results indicate that the inhibition of these post-prenylation-processing steps — particularly that of ICMT-catalysed methylation — might provide a better approach to the control of cancer-cell proliferation 8.
STE14 gene is required for the methylation of the C-terminal farnesyl cysteine residue in the production of active a factorand other polypeptides 3.
Lipidation reaction, farnesylation of the cysteine residue in the peptide is required for the methyltransferase activity, suggesting that methyl esterification follows the lipidation reaction in the cell 3.
Insulin secretion, a membrane-associated activity that methylesterifies prenylcysteine has been detected in S. cerevisiae and mammals, and a single activity appears to methylesterify both farnesylcysteine and geranylgeranyl cysteine. A link between receptor-mediated signal transduction and carboxylmethylation of Ras superfamily proteins has been suggested to exist in human neutrophils, and carboxylmethylation of Rap1, a member of the family, has been demonstrated in regulated insulin secretion 9.
1. Fujiyama A, Tsunasawa S, Tamanoi F, Sakiyama F (1991). S-Farnesylation and methyl esterification of c-terminal domain of yeast RASB protein prior to fatty acid acylation. JBC., 266(27): 17926-17931.
2. Imai Y, Davey J, Kawagishi-Kobayashi M, Yamamoto M (1997). Genes encoding farnesyl cysteine carboxyl methyltransferase in schizosaccharomyces pombe and xenopus laevis. Molecular and celluar biology.,17(3): 1543–1551.
3. Hrycyna CA, Clarke S (1990). Farnesyl cysteine c-terminal methyltransferase activity is dependent upon the ste14 gene product in Saccharomyces cerevisiae. Molecular and cellular Biology, 10(10):5071-5076.
4. Anderegg RJ, Betz R, Carr SA, Crabb JW, Duntze W (1988). Structure of Saccharomyces cerevisiae mating hormone a-factor. J. Biol. Chem., 263:18236–18240.
5. Clarke S (1992). Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu. Rev. Biochem., 61:355–386.
6. Schafer WR,Rine J (1992). Protein prenylation: genes, enzymes, targets, and functions. Annu. Rev. Genet., 30:209–237.
7. Sinensky M, Lutz R (1992). The prenylation of proteins. BioEssays., 14:25–31.
8. Ann M (2005). Post-prenylation-processing enzymes as new targets in oncogenesis. Nature Reviews Cancer., 5:405–412.
9. Leiser M, Efrat S, Fleischer N (1995). Evidence that Rap1 carboxylmethylation is involved in regulated insulin secretion. Endocrinology, 136:2521–2530.
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