Association of oncogenic ras proteins with cellular membranes appears to be a crucial step in transformation. ras is synthesized as a cytosolic precursor, which is processed to a mature form that localizes to the plasma membrane. This processing involves, in part, a conserved sequence, Cys-Ali-Ali-Xaa (in which Ali is an amino acid with an aliphatic side chain and Xaa is any amino acid), at the COOH terminus of ras proteins. Yeast a-factor mating hormone precursor also possesses a COOH-terminal Cys-Ali-AliXaa sequence. However, while the COOH-terminal cysteine has been implicated as a site of palmitoylation of ras proteins, in mature a-type mating factor this residue is modified by an

p6O0, the transforming protein kinase of Rous sarcoma virus, contains the 14-carbon saturated fatty acid, myristic acid, linked through an amide bond to the a-amino group of its NH2-terminal glycine residue. Myristic acid is known to be attached to four other eukaryotic proteins. In each case the fatty acid is also linked through an amide bond to an NH2-terminal glycine. We have used oligonucleotidedirected mutagenesis to examine the amino acid specificity of the enzyme that myristoylates the NH2 terminus of these proteins. Replacement of the NH2-terminal glycine in p6O1 with either alanine or glutamic acid prevented myristoylation completely. This indicates that the myristoylating enzyme may have an absolute specificity for glycine. Strikingly, neither nonmyristoylated mutant src protein induced morphological transformation of infected cells, even though wild-type levels of phosphorylation of cellular proteins on tyrosine were observed in these cells. Since conversion of the NH2-terminal residue from glycine to alanine should have little effect on the conformation of p6O1, the inability of this mutant p6Owr protein to induce morphological transformation suggests that the myristoyl moiety is essential for the transforming activity of the protein.Cellular transformation by Rous sarcoma virus results from the expression of a single viral protein designated p6011 (1). p60src functions as a tyrosine-specific protein kinase (2, 3) in vivo and is also reported to phosphorylate phosphatidylinositol in vitro (4). Tyrosine phosphorylation may be crucial in the control of cellular proliferation. The mitogens epidermal growth factor (5), platelet-derived growth factor (6), and insulin-like growth factor (7) all stimulate tyrosine protein kinase activity when they bind to their cell-surface receptors. Immunofluorescence (8), immunoelectron microscopy (9), and cell fractionation (10-12) all suggest that a significant fraction of the p60'" in transformed cells is associated with the cytoplasmic face of the plasma membrane. p60'1 may therefore deliver an unregulated mitogenic signal through the continuous phosphorylation ofone or more proteins involved in the normal regulation of proliferation.p60src is bound firmly to cellular membranes yet contains no large cluster of hydrophobic amino acids similar to those which are responsible for anchoring membrane-bound proteins such as the HLA and H-2 glycoproteins (human and murine major histocompatibility proteins) to a lipid bilayer. p60s' does, however, contain covalently bound myristic acid, a rare 14-carbon saturated fatty acid (13). This myristic acid moiety is attached by an amide linkage to the a-amino group of the NH2-terminal glycine residue of p6src (14). Consequently, an attractive hypothesis is that the hydrophobic myristoyl group plays a role in binding p6OSrc to membranes.Myristoylation is an uncommon form of protein modification. Nevertheless, the amino acid to which myristic acid is attached has been identified unambiguously in four additional proteins. The c...

We have introduced a variety of amino acid substitutions into carboxyl-terminal CAIA2X sequence (C = cysteine; A = aliphatic; X = any amino acid) of the oncogenic[Vall2]Ki-Ras4B protein to identify the amino acids that permit Ras processing (isoprenylation, proteolysis, and carboxyl methylation), membrane association, and transformation in cultured mammalian cells. While all substitutions were tolerated at the Al position, substitutions at A2 and X reduced transforming activity. The A2 residue was important for both isoprenylation and AAX proteolysis, whereas the X residue dictated the extent and specificity of isoprenoid modification only. Differences were observed between Ras processing in living cells and farnesylation efficiency in a cell-free system. Finally, one farnesylated mutant did not undergo either proteolysis or carboxyl methylation but still displayed efficient membrane association (==50%) and transforming activity, indicating that farnesylation alone can support Ras transforming activity. Since both farnesylation and carboxyl methylation are critical for yeast a-factor biological activity, the three CAAXsignaled modifications may have different contributions to the function of different CAAX-containing proteins.An association with the plasma membrane is critical for Ras transforming activity (1-3), and this association is promoted by a series of three closely linked posttranslational modification steps signaled by the consensus carboxyl-terminal CA1A2X motif (C = cysteine; A = any aliphatic amino acid; X = any amino acid) present in all Ras proteins. Addition of the 15-carbon farnesyl isoprenoid to the cysteine of the CAAX sequence is followed by proteolytic removal of the AAX residues and carboxyl methylation of the now terminal cysteine residue. Mutant Ras proteins lacking either the cysteine or the AAX residues are completely blocked in processing and are cytosolic and completely nontransforming. Posttranslational processing is critical for Ras function, but the precise contribution of each of the three CAAXsignaled processing steps to Ras membrane association and transforming activity remains to be established. The enzymes responsible for Ras processing are now beginning to be characterized. A cytosolic Ras farnesyltransferase activity, identified in both mammalian (4-6) and yeast (7, 8) cells, requires recognition of only the CAAX sequence to farnesylate the cysteine residue. In contrast to farnesyltransferase, the enzymatic activities for the AAX proteolysis (9) and carboxyl methylation (9-12) steps have been detected in the membrane component offractionated cells and tissues.In vitro studies with both synthetic peptides and chimeric Ras proteins have provided details of the sequence requirements for Ras farnesyltransferase modification. The residue at the A1 position can vary, while a much more restricted set of A2 and X residues permits efficient isoprenoid modification (13-15). The X residue also specifies whether the protein is modified by a farnesyl or by a geranylgeranyl group ...

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...