Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation.

Abstract

Oncogenic forms of ras proteins are synthesized in the cytosol and must become membrane associated to cause malignant transformation. Palmitic acid and an isoprenoid (farnesol) intermediate in cholesterol biosynthesis are attached to separate cysteine residues near the C termini of H-ras, N-ras, and Kirsten-ras (K-ras) exon 4A-encoded proteins. These lipid modifications have been suggested to promote or stabilize the association of ras proteins with membranes. Because preventing isoprenylation also prevents palmitoylation, examining the importance of isoprenylation alone has not been possible. However, the oncogenic human protein is not palmitoylated but is isoprenylated, membrane associated, and fully transforming. We therefore constructed mutant [Val'2]K-ras 4B proteins that were not isoprenylated to examine the effects of isoprenylation in the absence of palmitoylation. The nonisoprenylated mutant proteins both failed to associate with membranes and did not transform NIH 3T3 cells. In addition, inhibition of isoprenoid and cholesterol synthesis with the drug compactin also decreased [Val'2]K-ras 4B protein isoprenylation and membrane association. These results unequivocally demonstrate that isoprenylation, rather than palmitoylation, is essential for ras membrane binding and ras transforming activity. These rmdings clearly indicate the biological significance of ras protein modification by farnesol and suggest that this modification may be important for facilitating the processing, trafficking, and biological activity of other isoprenylated proteins.''Because K-ras is the most frequently activated oncogene in a wide spectrum of human malignancies, study of-this pathway could lead to important therapeutic treatments.The three cellular ras genes (H-, N-, and K-ras) encode related 21-kDa guanine nucleotide-binding proteins (GDP and GTP) (1). The biologic function of normal ras proteins is unknown. However, activated ras proteins, containing substitutions at residues 12, 13, or 61, can malignantly transform cells and are frequently detected in a wide spectrum of human neoplasms (1). Alterations in GTPase or GTP-binding properties of oncogenic ras proteins due to these activating substitutions favor formation ofthe active, GTP-ras complex (2-4).The ras proteins are synthesized in the cytosol as inactive precursors and must undergo a series of posttranslational modifications to become membrane-associated and biologically active (5-9). The four C-terminal amino acids of ras proteins comprise a consensus sequence, CAAX, in which C represents cysteine, A represents any aliphatic amino acid, and X represents any amino acid; this motif is believed to signal the posttranslational modifications of ras proteins.Specifically, these posttranslational modifications include (i) removal of the three C-terminal amino acid residues, (ii) carboxyl methylation of the C-terminal cysteine, (iii) attachment of palmitic acid to a cysteine residue(s) near the C terminus, and (iv) attachment of an isoprenoid intermediate in c...

Ras proteins: paradigms for compartmentalised and isoform-specific signalling

“…Mutagenesis of 4 or more lysines in the K(B)-Ras hexalysine stretch or the palmitoylated cysteines in H-Ras inhibits the ability of Ras to stably interact with membranes [44]. Similarly, mutation of the farnesylated cysteine prevents subsequent post-translational modifications and renders Ras cytosolic, inhibiting the potential for Ras activation [45]. For this reason, farnesyl transferase inhibitors were promoted as potential anti-Ras chemotherapeutic drugs however their effectiveness was undermined by the ability of both K(B)-Ras and N-Ras to be alternatively prenylated by geranylgeranyl transferase [46].…”

Stimulated human leukocytes cause activating mutations in the K-ras proto-oncogene

“…1 mg of leukocyte-treated K-ras 4B or H-ras plasmid DNA, 1 mg of purine and xanthine oxidase-treated K-ras 4B plasmid DNA, or 400 ng of 8 hydroxydeoxyguanosinecontaining v-H-ras/K-ras 4B hybrid plasmid DNA was transfected, along with 20 mg of carrier calf thymus DNA, into NIH3T3 cells by calcium phosphate precipitation, according to previously described methods (Jackson et al, 1990). Transfected dishes were maintained in Dulbecco's modi®ed Eagle's media/10% calf serum for 14 days, and subsequently analysed for the presence of morphologically transformed foci.…”

“…Subcloned secondary ras transformants were labeled overnight with 200 mCi/ml of 35 S Met/Cys (Trans 35 Slabel, ICN), and proteins contained in these cells were immunoprecipitated with anti-Ras antibody, Y13-259 (Furth et al, 1982), and analysed by SDS ± PAGE and uorography, as previously described (Jackson et al, 1990).…”

RhoA Prenylation Is Required for Promotion of Cell Growth and Transformation and Cytoskeleton Organization but Not for Induction of Serum Response Element Transcription

“…Protein prenylation is important in targeting proteins to cellular membranes but also in protein-protein interactions (18 -20). This process appeared to be critical for the oncoprotein Ras functions as observed with the dependence of its transforming activity on prenylation (21,22). Hence, over the past decade the functional role of protein prenylation has been intensively studied for Ras, while a few studies took interest in the other proteins.…”