Activation of a human c-K-ras oncogene

The human lung carcinomas PR310 and PR371 contain activated c-K-ras oncogenes. The oncogene of PR371 was found to present a mutation at codon 12 of the first coding exon which substitutes cysteine for glycine in the encoded p21 protein. We report here that the transforming gene of PR310 tumor contains a mutation in the second coding exon. An A----T transversion at codon 61 results in the incorporation of histidine instead of glutamine in the c-K-ras gene product. By constructing c-K-ras/c-H-ras chimeric genes we show that this point mutation is sufficient to confer transforming potential to ras genes, and that a hybrid ras gene coding for a protein mutant at both codons 12 and 61 is also capable of transforming NIH3T3 cells. The relative transforming potency of p21 proteins encoded by ras genes mutant at codons 12, 61 or both has been analyzed. Our studies also show that the coding exons of ras genes, including the fourth, can be interchanged and the chimeric p21 ras proteins retain their oncogenic ability in normal rodent established cell lines.     

Malignant transformation of murine fibroblasts by a human c-Ha-ras-1 oncogene does not require a functional epidermal growth factor receptor.

Although mutations in ras genes are thought to be important for the development of about 20% of human tumors, almost nothing is known about the way in which these mutations lead to cellular transformation. The known biochemical properties of the 21-kilodalton ras proteins suggest that they may behave as G proteins, regulating the proliferation of cells in response to growth factor stimulation of a receptor. Although the putative receptor(s) has not been identified, several lines of evidence, in particular the fact that rodent cell lines containing ras oncogenes produce transforming growth factor alpha, have suggested that the epidermal growth factor (EGF) receptor is involved in ras transformation. Here we show that murine fibroblasts with no EGF receptors can be transformed to a completely malignant phenotype with a mutated ras gene. It appears, therefore, that the EGF receptor is not required for ras-mediated transformation of these cells.     

Activation of ras genes in human tumors does not affect localization, modification, or nucleotide binding properties of p21

A comparison of proteins encoded by normal human ras genes and by mutant rasH or rasK genes activated in human carcinomas revealed no changes in subcellular localization, posttranslational modification, or guanine nucleotide binding associated with activation. Subcellular fractionation indicated that both normal and activated ras proteins were associated exclusively with the membrane fraction. Furthermore, both normal and activated ras proteins exhibited similar degrees of posttranslational acylation. The KD for dGTP binding was 1.0-2.2 X 10(-8) M, with no consistent differences between normal and activated ras proteins. In addition, a survey of 13 possible competing nucleotides revealed no differences in the specificity of nucleotide binding associated with ras gene activation. These results indicate that structural mutations which activate ras gene transforming activity do not alter the protein's known biochemical parameters and in particular do not affect the protein's intrinsic ability to bind guanine nucleotides.     

The ras superfamily proteins

Several recent discoveries indicate that the ras genes, frequently activated to a transforming potential in some human tumours, belong to a large family that can be divided into three main branches: the first branch represented by the ras, ral and rap genes; the second branch, by the rho genes; and the third branch, by the rab genes. The C-terminal end of the encoded proteins always includes a cystein, which may become fatty-acylated, suggesting a sub-membrane localization. The ras superfamily proteins share four regions of high homology corresponding to the GTP binding site; however, even in these regions, significant differences are found, suggesting that the various proteins may possess slightly different biochemical properties. Recent reports show that some of these proteins play an essential role in the control of physical processes such as cell motility, membrane ruffling, endocytosis and exocytosis. Nevertheless, the characterization of the proteins directly interacting with the ras or ras-related gene-products will be required to precisely understand their function.     

H-ras activation in benign and self-regressing skin tumors (keratoacanthomas) in both humans and an animal model system.

The involvement of the ras oncogenes in tumorigenesis was investigated in keratoacanthomas, which are benign and self-regressing skin tumors, both in humans and in a corresponding animal model system. Keratoacanthomas were induced on rabbit ears by repeated applications of 7,12-dimethylbenz(a)anthracene. About 60% of the tumor DNAs produced transformed foci after transfection into NIH 3T3 cells, and in all of them the transforming gene was identified as H-ras by Southern and Northern (RNA) hybridization. Immunoprecipitation experiments suggested that the transforming rabbit H-ras protein carried a mutation in codon 61. In addition, an activated H-ras gene was detected in a human keratoacanthoma by using a nude mouse tumorigenesis assay after transfection of tumor DNA into NIH 3T3 cells. This is the first report of ras activation in a benign human tumor. The transforming human H-ras gene showed a point mutation in codon 61 that would result in leucine instead of the glutamine present in the normal gene product. The finding of ras activation in tumors that are not only benign but also self-regressing indicates that activated ras genes are not sufficient to maintain a neoplastic phenotype, although they likely play a role in early stages of tumorigenesis.     

Human papillomavirus type 16 DNA cooperates with activated ras in transforming primary cells.

The close association of human papillomavirus type 16 DNA with a majority of cervical carcinomas implies some role for the virus in this type of cancer. To define the transforming properties of HPV-16 DNA in vitro we have now performed transfection experiments on baby rat kidney cells using HPV-16 DNA in conjunction with an activated ras gene. We have demonstrated that a 6.6-kb DNA fragment, containing the early genes of HPV-16 under the control of Moloney murine leukaemia virus long terminal repeats (MoMuLV-LTRs), cooperates with EJ-ras in transforming these cells. Both DNAs are required and neither alone is effective. The cooperating activity appears to reside in a protein or proteins derived from the E6/E7 region of the HPV-16 genome.     

Structural and functional properties of ras proteins

The ras proteins belong to a family of related polypeptides that are present in all eukaryotic organisms from yeast to human. Their extraordinary evolutionary conservation suggests that they have essential cellular functions, although their exact role remains unknown. Mutations in specific amino acids and overexpression of normal proteins have been linked to altered proliferation and/or differentiation and, particularly, to neoplastic processes. Mature ras proteins are located on the inner side of the plasma membrane, and their biochemical properties include binding and exchange of guanine nucleotides and GTPase activity. The favored hypothesis for ras function is that these proteins exist in an equilibrium between an inactive conformation (p21.GDP) and an active conformation (p21. GTP) in which they are able to interact with their as yet unknown cellular target or targets. Similarities in cellular location, structure, and biochemistry with other known regulatory (G) proteins suggest that they play a role in transduction of signals from the cell surface. The elucidation of the crystal structure of normal and transforming ras proteins and the identification of cellular proteins that interact directly with them (GAP, CDC25) or suppress some of their biological effects (Krev-1) have opened new avenues in the search for their elusive cellular targets and in the elucidation of the functional role of ras gene products.     

Activation of cellular p21ras by myristoylation

p21ras is palmitoylated on a cysteine residue near the C-terminus. Changing Cys-186 to Ser in oncogenic forms produces a non-palmitoylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. To examine whether palmitate acts in a general way to increase ras protein hydrophobicity, or is involved in more specific interactions between p21ras and membranes, we constructed genes that encode non-palmitoylated ras proteins containing myristic acid at their N-termini. Myristoylated, activated ras, without palmitate (61Leu/186Ser) exhibited both efficient membrane association and full transforming activity. Unexpectedly, we found that myristoylated forms of normal cellular ras were also potently transforming. Myristoylated c-ras retained the high GTP binding and GTPase characteristic of the cellular protein and, moreover, bound predominantly GDP in vivo. This implied that it continued to interact with GAP (GTPase-activating protein). While the membrane binding induced by myristate permitted transformation, only palmitate produced a normal (non-transforming) association of ras with membranes and must therefore regulate ras function by some unique property that myristate does not mimic. Myristoylation thus represents a novel mechanism by which the ras proto-oncogene protein can become transforming.     

Regional chromosomal localization of N-ras, K-ras-1, K-ras-2 and myb oncogenes in human cells

The identification of transforming genes in human tumor cells has been made possible by DNA mediated gene transfer techniques. To date, it has been possible to show that most of these transforming genes are activated cellular analogues of the ras oncogene family. To better understand the relationship between these oncogenes and other human genes, we have determined their chromosomal localization by analyzing human rodent somatic cell hybrids with molecularly cloned human proto-oncogene probes. It was possible to assign N-ras to chromosome 1 and regionally localize c-K-ras-1 and c-K-ras-2 to human chromosomes 6pter-q13 and 12q, respectively. These results along with previous studies demonstrate the highly dispersed nature of ras genes in the human genome. Previous reports indicated that the c-myb gene also resides on chromosome 6. It has been possible to sublocalize c-myb to the long arm of chromosome 6 (q15-q21). The non-random aberrations in chromosomes 1, 6 and 12 that occur in certain human tumors suggest possible etiologic involvement of ras and/or myb oncogenes in such tumors.     

Reversal of transformed phenotype by monoclonal antibodies against Ha-ras p21 proteins

The transforming activities of p21 ras proteins have been determined by micro-injection of these proteins into NIH3T3 cells. In order to facilitate functional studies on the effect of ras proteins on malignant transformation and normal cellular growth, analysis has been made with three monoclonal antibodies (YA6-172, Y13-238 and Y13-259) as originally reported by Furth et al. (J virol 43 (1982) 294). Purified immunoglobulin of Y13-259 has the highest titer of binding to bacterially synthesized p21 ras proteins. Experimental analyses indicate that only Y13-259 antibody will neutralize the transforming activity of the co-injected bacterially synthesized ras protein and the neutralization effect was blocked by co-injection of excess ras protein. In addition, micro-injection of Y13-259 immunoglobulin into transformed NIH3T3 cells (obtained by DNA transfection of NIH3T3 cells with molecularly cloned ras gene) reversed their transformed phenotypes. These results indicate that both bacterially synthesized p21 ras proteins and the natural ras proteins produced in NIH3T3 cells were neutralized by Y13-259 antibody.     

Genetic analysis of yeast RAS1 and RAS2 genes

We present a genetic analysis of RAS1 and RAS2 of S. cerevisiae, two genes that are highly homologous to mammalian ras genes. By constructing in vitro ras genes disrupted by selectable genes and introducing these by gene replacement into the respective ras loci, we have determined that neither RAS1 nor RAS2 are by themselves essential genes. However, ras1 - ras2 - spores of doubly heterozygous diploids are incapable of resuming vegetative growth. We have determined that RAS1 is located on chromosome XV, 7 cM from ade2 and 63 cM from his3; and RAS2 is located on chromosome XIV, 2 cM from met4 . We have also constructed by site-directed mutagenesis a missense mutant, RAS2val19 , which encodes valine in place of glycine at the nineteenth amino acid position, the same sort of missense mutation that is found in some transforming alleles of mammalian ras genes. Diploid yeast cells that contain this mutation are incapable of sporulating efficiently, even when they contain wild-type alleles.     

Role of a ras homolog in the life cycle of Schizosaccharomyces pombe

We have analyzed the function of the only ras homolog in S. pombe detectable by Southern blotting, ras1, which is homologous to mammalian ras genes and has been cloned. We have disrupted the ras1 gene and have replaced it with ras1Val17, which corresponds to a transforming variant of mammalian ras. Loss of ras1 activity by disruption results in the complete inability to mate. The cell body of a ras1- strain is extensively deformed, and a ras1-/ras1- diploid sporulates very poorly. Unlike RAS1 and RAS2 of S. cerevisiae, ras1 of S. pombe appears to have no effect on adenylate cyclase activity. This suggests that the target enzymes presumably modulated by ras proteins in signal transduction are not the same for all organisms.     

Saccharomyces cerevisiae synthesizes proteins related to the p21 gene product of ras genes found in mammals.

A family of normal vertebrate genes and oncogenes has been called the ras gene family. The name ras was assigned to this gene family based on the species of origin of the viral oncogenes of the rat-derived Harvey and Kirsten murine sarcoma viruses. There are now three known functional members of the ras gene family, and genes homologous to ras genes have been detected in the DNA of a wide variety of mammals and in Drosophila melanogaster. Prior experiments have detected proteins coded for by ras genes in a large number of normal cells, cell lines, and tumors. We report here the detection of ras-related proteins in D. melanogaster, a result predicted by the earlier detection of ras-related genes in the Drosophila genome. We also report for the first time the detection of ras-related proteins in a single-cell eucaryocyte, Saccharomyces cerevisiae. These proteins, approximately 30K in size, are recognized by both a monoclonal antibody which binds to the p21 coded for by mammalian ras genes and a polyclonal rat serum made by transplanting a v-Ha-ras-induced tumor in Osborne-Mendel rats. The p21 of v-Ha-ras and the 30K proteins from S. cerevisiae share methionine-labeled peptides as detected by two-dimensional tryptic peptide maps. The results indicate that S. cerevisiae synthesizes ras-related proteins. A genetic analysis of the function of these proteins for yeast cells may now be possible.     

Transformation by the k-ras oncogene correlates with increases in phospholipase A2 activity, glycerophosphoinositol production and phosphoinositide synthesis in thyroid cells.

Two cell lines transformed by the k-ras oncogene (KiKi and KiMol cells) and a temperature sensitive clone (Ts), all originated from a normal rat thyroid line (FRTL5 cells), have been employed to analyse the intracellular mechanisms affected by the ras p21. In k-ras transformed cells two phosphoinositide derivatives, glycerophosphoinositol and inositol monophosphate, were markedly increased, whereas inositol bisphosphate and trisphosphate maintained the same level as in normal cells. Cytosolic Ca2+ was also unaffected. This indicates that in epithelial cells the phospholipase C activity is not altered upon ras transformation. The formation of glycerophosphoinositol involved the activation of a phosphoinositide specific phospholipase A2. The higher phospholipase A2 activity in ras transformed cells could be further demonstrated by the increase in total arachidonic acid release. In the Ts clone the increase in glycerophosphoinositol and inositol monophosphate was evident only at the permissive temperature (33 degrees C), whereas it disappeared at 39 degrees C. At 33 degrees C the cells were also characterized by an enriched membrane pool of phosphoinositides. All these changes occurred in parallel with morphological transformation. We propose that cell transformation by the k-ras oncogene affects different steps of the membrane lipid metabolism, among which the most prominent one is the activation of a phosphoinositide specific phospholipase A2. These effects could originate mitogenic metabolites. Moreover, they correlate well with the induction of the malignant phenotype.     

Crystal structures at 2.2 A resolution of the catalytic domains of normal ras protein and an oncogenic mutant complexed with GDP

The biological functions of ras proteins are controlled by the bound guanine nucleotide GDP or GTP. The GTP-bound conformation is biologically active, and is rapidly deactivated to the GDP-bound conformation through interaction with GAP (GTPase Activating Protein). Most transforming mutants of ras proteins have drastically reduced GTP hydrolysis rates even in the presence of GAP. The crystal structures of the GDP complexes of ras proteins at 2.2 A resolution reveal the detailed interaction between the ras proteins and the GDP molecule. All the currently known transforming mutation positions are clustered around the bound guanine nucleotide molecule. The presumed "effector" region and the GAP recognition region are both highly exposed. No significant structural differences were found between the GDP complexes of normal ras protein and the oncogenic mutant with valine at position 12, except the side-chain of the valine residue. However, comparison with GTP-analog complexes of ras proteins suggests that the valine side-chain may inhibit GTP hydrolysis in two possible ways: (1) interacting directly with the gamma-phosphate and altering its orientation or the conformation of protein residues around the phosphates; and/or (2) preventing either the departure of gamma-phosphate on GTP hydrolysis or the entrance of a nucleophilic group to attack the gamma-phosphate. The structural similarity between ras protein and the bacterial elongation factor Tu suggests that their common structural motif might be conserved for other guanine nucleotide binding proteins.     

Oncogenic genes and human malignancy.

All vertebrates possess a series of genes which are homologs of the oncogenic genes of acute transforming retroviruses. Two lines of evidence suggest that these genes may play a role in the development of human malignancy: (1) DNA from a variety of human tumors transforms NIH 3T3 mouse fibroblasts and the transforming genes from a number of carcinomas, sarcomas, and hematological malignancies have been identified as members of a family of genes, the ras family, closely related to the oncogenic genes of the Harvey and Kirsten murine sarcoma viruses; and (2) correlations exist between the chromosomal localizations of certain oncogenes and the chromosomal breakpoints in specific translocations and deletions in certain human malignancies. In three separate hematological malignancies, alterations in more than one oncogenic gene may be involved in the neoplastic process.     

Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus.

Previous studies of premature chain termination mutants and in frame deletion mutants of the p21 ras transforming protein encoded by the transforming gene of Harvey murine sarcoma virus (Ha-MuSV) have suggested that the C terminus is required for cellular transformation, lipid binding, and membrane localization. We have now further characterized the post-translational processing of these mutants and have also studied two C-terminal v-rasH point mutants: one encodes serine in place of cysteine-186, the other threonine for valine-187. The Thr-187 mutant was transformation-competent, and its p21 protein was processed normally, as was the p21 encoded by a transformation-competent deletion mutant from which amino acids 166-175 had been deleted. The Ser-186 mutant was defective for transformation. The p21s encoded by the Ser-186 mutant and by the previously described transformation-defective mutants did not undergo the posttranslational processing common to biologically active ras proteins: their electrophoretic migration rate did not change, they remained in the cytosol, and they failed to bind lipid. Since the cell-encoded ras proteins also contain this cysteine, we conclude that this amino acid residue is required for all ras proteins.     

The oncogenic forms of N-ras or H-ras prevent skeletal myoblast differentiation.

Differentiation of skeletal muscle involves withdrawal of myoblasts from the cell cycle, fusion to form myotubes, and the coordinate expression of a variety of muscle-specific gene products. Fibroblast growth factor and type beta transforming growth factor specifically inhibit myogenesis; however, the transmembrane signaling pathways responsible for suppression of differentiation by these growth factors remain elusive. Because ras proteins have been implicated in the transduction of growth factor signals across the plasma membrane, we used DNA-mediated gene transfer to investigate the potential involvement of this family of regulatory proteins in the control of myogenesis. Transfection of the mouse skeletal muscle cell line C2 with the oncogenic forms of H-ras or N-ras completely suppressed both myoblast fusion and induction of the muscle-specific gene products nicotinic acetylcholine receptor and creatine kinase. Inhibition of differentiation by activated ras genes occurred at the level of muscle-specific mRNA accumulation. In contrast, proto-oncogenic forms of N-ras or H-ras had no apparent effects on the ability of C2 cells to differentiate. Myoblasts transfected with activated ras genes exhibited normal growth properties and ceased proliferating in the absence of mitogens, indicating that ras inhibited differentiation through a mechanism independent of cell proliferation. These results demonstrate that activated ras gene products mimic the inhibitory effects of fibroblast growth factor and type beta transforming growth factor on myogenic differentiation and suggest that each of these regulators of myogenesis may operate through a common intracellular pathway.