Human cells contain four homologous Ras proteins, but it is unknown whether each of these Ras proteins participates in distinct signal transduction cascades or has different biological functions. To directly address these issues, we assessed the relative ability of constitutively active (G12V) versions of each of the four Ras homologs to activate the effector protein Raf-1 in vivo. In addition, we compared their relative abilities to induce transformed foci, enable anchorage-independent growth, and stimulate cell migration. We found a distinct hierarchy between the four Ras homologs in each of the parameters studied. The hierarchies were as follows: for Raf-1 activation, Ki-Ras 4B > Ki-Ras 4A >>> N-Ras > Ha-Ras; for focus formation, Ha-Ras >/= Ki-Ras 4A >>> N-Ras = Ki-Ras 4B; for anchorage-independent growth, Ki-Ras 4A >/= N-Ras >>> Ki-Ras 4B = Ha-Ras = no growth; and for cell migration, Ki-Ras 4B >>> Ha-Ras > N-Ras = Ki-Ras 4A = no migration. Our results indicate that the four Ras homologs significantly differ in their abilities to activate Raf-1 and induce distinctly different biological responses. These studies, in conjunction with our previous report that demonstrated that the Ras homologs can be differentially activated by upstream guanine nucleotide exchange factors (Jones, M. K., and Jackson, J. H. (1998) J. Biol. Chem. 273, 1782-1787), indicate that each of the four Ras proteins may qualitatively or quantitatively participate in distinct signaling cascades and have significantly different biological roles in vivo. Importantly, these studies also suggest for the first time that the distinct and likely cooperative biological functions of the Ki-ras-encoded Ki-Ras 4A and Ki-Ras 4B proteins may help explain why constitutively activating mutations of Ki-ras, but not N-ras or Ha-ras, are frequently detected in human carcinomas.

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...

In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H202 added extracellularly in micromolar concentrations (10-100 MM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H202 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H202 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase.Cytotoxic doses of HOCI, added to target cells, did not induce DNA strand breakage, and myeloperoxidase added extracellularly in the presence of an H202-generating system, prevented the formation of DNA strand breaks in proportion to its H202 degrading capacity.The studies also indicated that H202 formed hydroxyl radical (OH) intracellularly, which appeared to be the most likely free radical responsible for DNA damage: OH was detected in cells exposed to H202; the DNA base, deoxyguanosine, was hydroxylated in cells exposed to H202; and intracellular iron was essential for induction of DNA strand breaks.

HOC, which is produced by the action of myeloperoxidase during the respiratory burst of stimulated neutrophils, was used as a cytotoxic reagent in P388D1 cells. Low concentrations of HOCI (10-20 MAM) caused oxidation of plasma membrane sulfhydryls determined as decreased binding of iodoacetylated phycoerythrin. These same low concentrations of HOCI caused disturbance of various plasma membrane functions: they inactivated glucose and aminoisobutyric acid uptake, caused loss of cellular K+, and an increase in cell volume. It is likely that these changes were the consequence of plasma membrane SH-oxidation, since similar effects were observed with para-chloromercuriphenylsulfonate (pCMBS), a sulfhydryl reagent acting at the cell surface. Given in combination pCMBS and HOCI showed an additive effect.Higher doses of HOCI (> 50 1AM) led to general oxidation of -SH, methionine and tryptophan residues, and formation of protein carbonyls. HOCI-induced loss of ATP and undegraded NAD was closely followed by cell lysis. In contrast, NAD degradation and ATP depletion caused by H202 preceded cell death by several hours. Formation of DNA strand breaks, a major factor of H202-induced injury, was not observed with HOCI.Thus targets of HOCI were distinct from those of H202 with the exception of glyceraldehyde-3-phosphate dehydrogenase, which was inactivated by both oxidants. (J. Clin. Invest. 1990. 85:554-562.) hypochlorite toxicity-plasma membrane protein oxidation * sulfhydryls