No abstract

We have developed a strategy to isolate mutant ras genes encoding proteins defective in GTP binding. Random in vitro mutagenesis of a v-Harvey (Ha)-ras expression vector was followed by an in situ GTP-binding assay on lysed bacterial colonies. Single amino acid substitutions at ras codon 83, 119, or 144 decreased the affinity of p21 for GTP by a factor of 25-100 primarily as a consequence of increased rates of dissociation of GTP from p21. Nevertheless, these mutant genes induced transformation of NIH 3T3 cells with efficiencies comparable to wild-type v-Ha-ras. In transformed cells, mutant p2is were phosphorylated to a degree similar to that of wild-type v-Ha-ras p21, suggesting that a decrease in affinity by a factor of 100 did not prevent the mutant ras protein from binding GTP in vivo. These results are discussed with respect to the role of GTP in the regulation of p21 function.Cellular ras genes, Harvey (Ha)-ras, Kirsten (Ki)-ras, and N-ras, have been detected as active transforming genes in a variety of human tumors by transfection of NIH 3T3 cells. Activation has been found to be the consequence of single amino acid substitutions at either position 12, 13, or 61 (1, 2). The transforming genes of Harvey and Kirsten murine sarcoma viruses, derived from the cellular Ha-ras and Ki-ras genes, respectively, differ from their normal cellular homologues by amino acid substitutions at positions 12 and 59, each of which confers transforming activity upon the viral proteins (1,3). The only additional difference between the cellular and viral Ha-ras proteins is a single amino acid change at position 122.The ras genes encode proteins of 21,000 daltons, termed p21, that reside in the cytoplasmic face of the plasma membrane (4). In addition, p21 binds GTP and GDP with high affinity (5-7) and displays GTPase activity (8)(9)(10)(11). Some viral proteins also possess a GTP-dependent autokinase activity that phosphorylates the threonine at position 59 (12).Although the function of p21 is not known, its membrane localization and interactions with guanine nucleotides are reminiscent of the G proteins, which regulate hormonesensitive adenylate cyclase (13), and transducin, which regulates a cyclic GMP phosphodiesterase in response to light in the retinal rod outer segment (14). For transducin and G proteins, binding of GTP promotes their interaction with the enzymes they regulate (13,14). It is a reasonable hypothesis, therefore, that p21 also acts to transduce an extracellular signal to an as yet unidentified intracellular effector molecule and that GTP binding and hydrolysis are critically involved in the regulation of this process.Despite the presumed importance of GTP binding to ras function, the sites of GTP binding and the biological consequences of alterations in GTP binding have not been defined. Although amino acid substitutions at position 12 or 61 impair GTPase activity (8-11, 15, 16), they have no effect on GTP binding (7,16 (17) an episome containing the iQ allele of the i gene and the kanamycin-resistance...

Single amino acid substitutions were introduced into a region of the rasH protein (residues 116, 117, and 119) homologous to a variety of diverse GTP-binding proteins. Each of the mutant p21 proteins displayed a significant reduction (10-to 5,000-fold) in GTP binding affinity. Activated rasH proteins deficient in GTP binding were unaltered in their ability to morphologically transform NIH 3T3 cells.The human ras family consists of three known genes (rasH, rasK, and rasN) which have been identified as active transforming genes in a variety of human neoplasms (5). The cellular ras genes encode closely related proteins designated p2is. These plasma-membrane-associated proteins bind guanine nucleotides with high affinity (9,26,32) and display a low GTPase activity (11,19,22,34). Amino acid sequence comparisons have identified homologies between the ras proteins and other proteins which exhibit a high specific affinity for guanine nucleotides, including bacterial elongation and initiation factors, tubulin, and members of the G protein family (16,18). Of these regions of shared sequence homology, the region represented by ras amino acids 110 through 120 is the most striking. We therefore used sitedirected mutagenesis to evaluate the possible role of these ras sequences in guanine nucleotide binding.A comparison of amino acid sequences representing a variety of guanine-nucleotide-binding proteins is shown in Table 1. A consensus sequence of Asn-Lys-X-Asp (human ras residues 116 through 119) was found in all GTP-binding proteins examined. To directly evaluate the involvement of these amino acids in the binding of GTP, oligonucleotidedirected mutagenesis (37, 38) was used to introduce single amino acid substitutions into a cDNA clone of a human rasH gene whose transforming potential was activated by substitution of leucine for glutamine at position 61 (rasH ; 8). Seventeen-base synthetic oligonucleotides, synthesized by the modified triester method, were used to introduce single nucleotide substitutions into a M13mp8 clone containing the rasH cDNA sequence. The following three mutations were isolated: rasH(61-Leu, 116-His), in which codon 116 was changed from AAC (Asn) to CAC (His); rasH(61-Leu, 117-Glu), in which codon 117 was changed from AAG (Lys) to GAG (Glu); and rasH(61-Leu, 119-His), in which codon 119 was changed from GAC (Asp) to CAC (His). The nucleotide sequences of the mutant clones were verified by dideoxy sequencing (31).To evaluate the guanine-nucleotide-binding properties of the mutated rasH proteins, each rasH mutation was constructed into a bacterial expression vector pXVR (8a), which directs synthesis of authentic mammalian p21, and then introduced into Escherichia coli PR13-Q. Upon induction with isopropylthio-3-D-galactoside, approximately 30% of the total bacterial protein was represented by p21. Bacterium-expressed p2is were purified as previously described

Microinjection of Xenopus oocytes with ras protein (p21) was used to investigate the role of phospholipid metabolism in ras-induced meiotic maturation. Induction of meiosis by ras was compared with induction by progesterone, insulin, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Neomycin, which specifically binds to phosphatidylinositides and inhibits their metabolism, blocked meiotic maturation induced by ras or insulin but not by progesterone or TPA. In addition, p21 and TPA, but not insulin or progesterone, stimulated the incorporation of 32p; into oocyte lipids. ras protein specifically stimulated 32p incorporation into phosphatidylinositides, whereas both ras and TPA stimulated 32p incorporation into phosphatidylcholine and phosphatidylethanolamine. The stimulatory effect of p21 on phosphatidylinositide metabolism correlated with the dose response and kinetics of ras-induced meiotic maturation. In addition, the ras oncogene protein was more potent than the proto-oncogene protein both in inducing meiotic maturation and in stimulating phosphatidylinositide metabolism. These results indicate that phosphatidylinositide turnover is required for ras-induced meiosis and suggest that phosphatidylinositide-derived second messengers mediate the biological activity of ras in Xenopus oocytes.The ras proto-oncogene family is highly conserved in evolution, being present in organisms ranging from yeasts to humans (see reference 2 for a review). In mammals, this gene family consists of three members that encode closely related proteins of 21,000 daltons, designated p2ls. ras oncogenes activated by point mutations are found in a significant fraction of human and carcinogen-induced animal neoplasms, indicating their frequent contribution to tumor development (2). In addition, ras proto-oncogenes appear to be involved in the normal proliferation of both yeast (22,40) and mammalian (15, 31) cells and may also function in cell differentiation (3,21,33).The ras genes encode plasma membrane proteins that bind GTP and GDP with high affinity and possess GTP hydrolysis activity (2). Most mutations that activate ras transforming potential result in single-amino-acid substitutions that increase the fraction of p21 in the GTP-bound state, which appears to be the physiologically active form (2). The regulation of p21 by guanine nucleotide binding is similar to the regulation of G proteins (20), suggesting that ras proteins function as signal-transducing molecules that regulate the metabolism of intracellular second messengers.In Saccharomyces cerevisiae, ras proteins stimulate the activity of adenylate cyclase (8,42). In vertebrate cells, however, adenylate cyclase does not appear to be affected by p21 (5), and the physiologically relevant target(s) for ras function has not been established. However, alterations in phosphatidylinositol (PI) metabolism and increased levels of diacylglycerol (DAG) have been observed in ras-transformed cells, suggesting the possibility that p21 regulates the turnover of phospholipid-de...