A ras effector domain mutant which is temperature sensitive for cellular transformation: interactions with GTPase-activating protein and NF-1.

A series of v-rasH effector domain mutants were analyzed for their ability to transform rat 2 cells at either low or high temperatures. Three mutants were found to be significantly temperature sensitive: Ile-36 changed to Leu, Ser-39 changed to Cys (S39C), and Arg-41 changed to Leu. Of these, the codon 39 mutant (S39C) showed the greatest degree of temperature sensitivity. When the same mutation was analyzed in the proto-oncogene form of ras(c-rasH), this gene was also found to be temperature sensitive for transformation. Biochemical analysis of the proteins encoded by v-rasH(S39C) and c-rasH(S39C) demonstrated that the encoded p21ras proteins were stable and bound guanine nucleotides in vivo at permissive and nonpermissive temperatures. On the basis of these findings, it is likely that the temperature-sensitive phenotype results from an inability of the mutant (S39C) p21ras to interact properly with the ras target effector molecule(s) at the nonpermissive temperature. We therefore analyzed the interaction between the c-rasH(S39C) protein and the potential target molecules GTPase-activating protein (GAP) and the GAP-related domain of NF-1, on the basis of stimulation of the mutant p21ras GTPase activity by these molecules in vitro. Assays conducted across a range of temperatures revealed no temperature sensitivity for stimulation of the mutant protein, compared with that of authentic c-rasH protein. We conclude that for this mutant, there is a dissociation between the stimulation of p21ras GTPase activity by GAP and the GAP-related domain NF-1 and their potential target function. Our results are also consistent with the existence of a distinct, as-yet-unidentified effector for mammalian ras proteins.     

Photoaffinity labeling with GTP of viral p21 ras protein expressed in Escherichia coli

The v-ras oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton protein, p21, which mediates transformation produced by that virus. Previous work has shown that both p21v-rasH and the cellular homolog p21c-rasH appear to bind guanine nucleotides. We report here the expression in Escherichia coli of v-rasH to produce a biochemically active p21 fusion protein which retains both guanine nucleotide binding and autophosphorylating activity. Furthermore, direct interaction of this protein with GTP is unequivocally demonstrated by photoaffinity labeling it with [alpha-32P]GTP.     

Nucleotide sequence of the two rat cellular rasH genes.

We present the nucleotide sequence of the coding region of the rat c-rasH-1 gene and a partial sequence analysis of the rat c-rasH-2 gene. By comparing these sequences with the Harvey murine sarcoma virus ras gene, we predict that the p21 protein encoded by the Harvey virus differs from the cellular c-rasH-1-encoded p21 at only two amino acids; those at positions 12 and 59. Alterations at each of these positions may play a role in activating the viral p21 protein. The c-rasH-2 gene is likely to be a nonfunctional pseudogene because it lacks introns, cannot be activated to transform NIH 3T3 cells, and differs in sequence from both c-rasH-1 and v-rasH at several base pair positions.     

Interaction of ras oncogene product p21 with guanine nucleotides

The nucleotide exchange reaction was observed with purified ras oncogene product p21 overproduced in Escherichia coli (Hattori, S. et al. (1985) Mol. Cell Biol. 5, 1449-1455) under various conditions. (NH4)2SO4 increased the rate of dissociation of bound GDP from c-rasH and v-rasH p21. The dissociation kinetics were those of a first order reaction, and there was a linear relationship between the rate constant and the (NH4)2SO4 concentration. At any concentration of (NH4)2SO4, the exchange rate was faster with v-rasH p21 than that with c-rasH p21. EDTA and (NH4)2SO4 synergetically stimulated the dissociation reaction. Nucleotide-free p21 was prepared by gel filtration on Sephadex G-25 in the presence of 5 mM EDTA and 200 mM (NH4)2SO4 at room temperature. The free p21 was quite thermolabile, but the addition of GDP or GTP completely protected p21 from thermal inactivation. The dissociation constants for GDP and GTP were determined with free p21 to be 8.9 and 8.2 nM, respectively, for v-rasH p21, and 1.0 and 2.6 nM for c-rasH p21. In the presence of 200 mM (NH4)2SO4, these dissociation constants increased 3- to 12-fold.     

The bovine papillomavirus E5 oncogene can cooperate with ras: identification of p21 amino acids critical for transformation by c-rasH but not v-rasH.

We have previously used a series of insertion-deletion mutants of the mutationally activated v-rasH gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass, and D. R. Lowy, Mol. Cell. Biol. 6:2646-2654, 1986). To determine if some of these amino acids are more important for the biological activity of c-rasH, we have now tested many of the same insertion-deletion mutants in the c-rasH form for their ability to transform NIH 3T3 cells. Since the transforming activity of c-rasH is low, we have used cotransfection with the bovine papillomavirus (BPV) genome to develop a more sensitive transformation assay for c-rasH mutants. The increased sensitivity of the assay, which is seen both in focal transformation and in anchorage-independent growth, is mediated by cooperation between the BPV E5 gene and ras. E5-dependent cooperation was seen for v-rasH as well as for c-rasH, which suggests that the major effect of E5 was to increase the susceptibility of the cell to transformation to a given level of ras activity. The cooperation assay was used to test the potential importance, in c-rasH, of codons 93 to 108, 123 to 130, and 166 to 183, which were nonessential for v-rasH transformation. Relative to the respective transforming activity of wild-type c-rasH and v-rasH, mutants with lesions in codons 102 and 103 were significantly less active in their c-rasH forms than in their v-rasH forms. We conclude that a region including amino acids 102 and 103 encodes a function that is more critical to c-rasH than to v-rasH. Guanine nucleotide exchange is one function that is compatible with such a phenotype.     

Physiological correlation between nucleoside-diphosphate kinases and the 21-kDa guanine-nucleotide binding proteins copurified with the enzymes from the cell membrane fractions of Ehrlich ascites tumor cells

The physiological correlation between nucleoside-diphosphate kinases (NDP-kinases) and the 21-kDa guanine nucleotide-binding proteins (G1 and G2) which are copurified with the enzymes from the cell membrane fractions of Ehrlich ascites tumor cells has been biochemically investigated in vitro. We found that: incubation of the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinases (F-I and F-II) with one of the nucleoside 5'-diphosphates in the presence of 1 mM Mg2+ or 0.25 mM Ca2+ results in the rapid formation of nucleoside 5'-triphosphates without strict base specificity; GDP on the guanine nucleotide-binding proteins (G1, G2 and recombinant v-rasH p21) acts as a phosphate acceptor for the high-energy phosphates of the phosphoenzyme in the presence of 0.25 mM Ca2+; and [32P]GTP is preferentially formed from the 32P-labelled phosphoenzyme F-I and GDP-bound G1 or GDP-bound recombinant v-rasH p21 protein, even if any other nucleoside 5'-diphosphates are present in the reaction mixture. Although [32P]GTP formed was bound with the guanine nucleotide-binding proteins, it was immediately hydrolyzed by the proteins themselves in the presence of 5 mM Mg2+, but not in the presence of 0.25 mM Ca2+. Available evidence suggests that NDP-kinase may be responsible for the activation of the guanine nucleotide-binding proteins (G1, G2 and p21 proteins) through phosphate transfer by the enzyme.     

Mutational analysis of a ras catalytic domain.

We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.     

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.     

Expression and characterization of ras mRNAs from Saccharomyces cerevisiae.

The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation. We recently reported [DeFeo-Jones et al., Nature (London) 306:707-709, 1983] the cloning of two ras homologs from the yeast Saccharomyces cerevisiae. The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product. We have also found proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol. Cell. Biol. 4:23-29, 1984). In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes. We show that both ras genes are expressed in the wild-type cell. Furthermore, we demonstrate by in vitro translation of hybrid-selected RASsc1 mRNA and immunoprecipitation of the translation products that the cloned RASsc1 gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody. Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates. The structural and biochemical homologies between ras gene products of S. cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.     

Hormone-regulated expression of cellular rasH oncogene in mammary carcinomas in rats

Differential gene expression has been observed in hormone-dependent rat mammary carcinomas during their growth and regression. A 22K MW protein, a prominent in vitro translation product of the growing as compared to the regressing tumor, was identified as the c-rasH-21,000-dalton transforming protein (p21) using a monoclonal antibody that reacts specifically with Harvey-related p21 species. The amount of p21-translated protein sharply decreased in the translation products of the regressing tumors within 6 hours post ovariectomy or dibutyryl cyclic AMP treatment. The results show that an enhanced expression of the c-rasH oncogene is associated with hormone-dependent growth of mammary carcinomas in vivo and that suppression of this oncogene precedes the tumor regression induced by either hormone withdrawal or dibutyryl cyclic AMP treatment.     

Harvey murine sarcoma virus: influences of coding and noncoding sequences on cell transformation in vitro and oncogenicity in vivo.

The rat-derived Harvey murine sarcoma virus (Ha-MuSV) contains a transduced ras oncogene activated by two missense mutations and flanked by rat retroviruslike VL30 sequences. Ha-MuSV induces focal transformation of mouse NIH 3T3 cells in vitro and tumors (fibrosarcomas and splenic erythroleukemias) in newborn mice. We have used these two assays to study the contribution of coding and noncoding viral sequences to the biological activity of Ha-MuSV. A good correlation was found between the in vitro and in vivo assays. In several different isogenic Ha-MuSV variants, those with a rasH gene that had one or both of the Ha-MuSV missense mutations were much more active biologically than the corresponding proto-oncogene. A Ha-MuSV variant that encoded the proto-oncogene protein induced lymphoid leukemias (with thymomas), with a relatively long latent period, rather than the fibrosarcomas and erythroleukemias characteristic of Ha-MuSV with one or both missense mutations. A VL30-derived segment with enhancer activity was identified downstream from v-rasH. A mutant Ha-MuSV from which this 3' noncoding segment was deleted expressed lower levels of the wild-type viral protein, displayed impaired transforming activity in vitro, and induced lymphoid leukemias (with thymomas). 5' noncoding rat c-rasH sequences were found to increase the biological activity of the virus when substituted for the corresponding segment of v-rasH. We conclude that (i) the biological activity of Ha-MuSV can be influence significantly by noncoding sequences located outside the long terminal repeat as well as by coding sequences, (ii) VL30 sequences positively regulate the expression of v-rasH, (iii) relatively low biological levels of ras, whether resulting from low-level expression of wild type v-rasH or high-levels of ras proto-oncogene protein, induce a type of tumor that differs from tumors induced by high biological levels of ras, and (iv) the in vivo pathogenicity of the Ha-MuSV variants correlated with their transforming activity on NIH 3T3 cells.     

v-rasH expression confers hormone-independent in vitro growth to LNCaP prostate carcinoma cells

The LNCaP human prostate cancer cell line is dependent on androgen for in vitro growth. To discover genes that may be responsible for progression of prostate cancer from hormone dependence to hormone independence, we transfected LNCaP cells with expression vectors that contained either the v-rasH or c-rasH gene under the control of the cadmium (Cd2+)-inducible human metallothionein-IIA promoter. Numerous derivative cell lines were isolated which manifested inducible expression of rasH p21 protein when the cells were treated with Cd2+. None of the cell lines transfected with c-rasH were found to have an altered growth phenotype. Several derivative cell lines expressing inducible v-rasH manifested hormone-independent growth in culture when treated with 10(-7) M Cd2+ . Cd2+ induction of v-rasH p21 was also shown to increase anchorage-independent colony formation of the v-rasH-expressing cell lines tested. Expression of a dominant mutated oncogene can change the hormone-dependent growth phenotype of prostate cancer cells.     

Viral Harvey ras p21 expressed in Escherichia coli purifies as a binary one-to-one complex with GDP

The ras oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton protein, p21, which mediates transformation. Viral Harvey ras p21, cloned into Escherichia coli HB101 lambda/pRAS1, has been purified to homogeneity by a three-step procedure. The purified E. coli p21 can be bound by a monoclonal antibody to viral Harvey ras p21 and has an amino acid composition consistent with that predicted from its DNA sequence. Purified E. coli p21 has been shown by HPLC analysis on an ion-exchange column to contain near stoichiometric amounts of GDP. This noncovalently associated GDP is seen in the UV absorption spectrum of the purified protein. The noncovalently associated GDP acts as a competitive inhibitor of the interaction of added guanine nucleotides with p21.     

ralGDS family members interact with the effector loop of ras p21.

Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.     

Characterization of the phosphorylation sites and the surrounding amino acid sequences of the p21 transforming proteins coded for by the Harvey and Kirsten strains of murine sarcoma viruses

The transforming protein coded for by the onc gene (v-rasHa) of Harvey murine sarcoma virus (Ha-MuSV) is the 21,000-dalton protein (p21) which is the immediate agent responsible for the virus-induced malignant transformation of normal cells. The p21 proteins of Ha-MuSV and the closely related Kirsten murine sarcoma virus are heavily phosphorylated in vivo. In the partially purified Ha-MuSV p21, the protein shows a guanine nucleotide-binding activity and, in addition, a very unique autophosphorylating activity at a threonine residue using as phosphoryl donor GTP but not ATP. In the present study, we compared the tryptic peptide maps of the Ha-MuSV p21 phosphorylated in vivo and in vitro. The results show that the major phosphorylation site is identical. Since the GTP-specific phosphorylation is very unique and distinct from all other known protein kinases, the present observation suggests that the in vitro enzymatic activity is responsible for the p21 phosphorylation in vivo. We have analyzed the amino acid sequence surrounding the major phosphorylation site of the Ha-MuSV p21 by automated Edman degradations of the tryptic phosphopeptides. Threonine residue 59 from the initiator methionine residue 1 of the p21 protein is the phosphorylated amino acid residue, and the surrounding amino acid sequence is NH2...-Thr-Cys-Leu-Leu-Asp-Ile-Leu-Asp-Thr-Thr(P)-Gly-Gln-Glu-Glu-Tyr-...COOH. The p21 proteins of both the Ha-MuSV and the closely related Kirsten murine sarcoma virus share the same phosphopeptide. The amino acid sequence of the phosphorylation site is distinct from all other known protein kinases.     

Development and analysis of a transformation-defective mutant of Harvey murine sarcoma tk virus and its gene product.

The Harvey murine sarcoma virus has been cloned and induces focus formation on NIH 3T3 cells. Recombinants of this virus have been constructed which include the thymidine kinase gene of herpes simplex virus type 1 in a downstream linkage with the p21 ras gene of Harvey murine sarcoma virus. Harvey murine sarcoma tk virus rescued from cells transfected with this construct is both thymidine kinase positive and focus inducing in in vitro transmission studies. The hypoxanthine-aminopterin-thymidine selectability of the thymidine kinase gene carried by this virus has been exploited to develop three mutants defective in the p21 ras sequence. All three are focus negative and thymidine kinase positive when transmitted to suitable cells. Of these, only one encodes a p22 that is immunologically related to p21. This mutant has been used to explore the relationship between the known characteristics of p21 and cellular transformation. Data presented herein indicate that the p21 of Harvey murine sarcoma virus consists of at least two domains, one which specifies the guanine nucleotide-binding activity of p21 and the other which is involved in p21-membrane association in transformed cells.     

Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins

The protein products of the mammalian ras genes, p21ras, are regulatory guanine nucleotide binding proteins that are involved in the control of cell proliferation, though the exact biochemical processes regulated are unknown. Recently a cytoplasmic protein has been identified that interacts with and increases the GTPase activity of p21ras. It has been shown that this GTPase-activating protein, or GAP, interacts with the effector domain of ras, leading us and others to propose that GAP may be the target for regulation by p21ras. It has become apparent that ras is part of a much larger family of proteins, and at least 15 ras-related genes have now been identified in the mammalian genome. Each encodes a small (about 21 kDa) guanine nucleotide binding protein, but the functions of none of these regulatory molecules are known. We report here that mammalian cytoplasmic extracts contain GAP-like activity toward the products of two other ras-related genes, R-ras and rho. It appears that p23R-ras interacts with the same 125-kDa GAP protein as p21ras whereas p21rho interacts with a distinct 29-kDa protein, rho GAP.     

Abnormal regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis.

Tumor cell lines derived from malignant schwannomas removed from patients with neurofibromatosis type 1 (NF1) have been examined for the level of expression of NF1 protein. All three NF1 lines examined expressed lower levels of NF1 protein than control cells, and the level in one line was barely detectable. The tumor lines expressed normal levels of p120GAP and p21ras. Although the p21ras proteins isolated from the tumor cells had normal (nonmutant) biochemical properties in vitro, they displayed elevated levels of bound GTP in vivo. The level of total cellular GAP-like activity was reduced in extracts from the tumor line that expresses very little NF1 protein. Introduction of the catalytic region of GAP into this line resulted in morphological reversion and lower in vivo GTP binding by endogenous p21ras. These data implicate NF1 protein as a tumor suppressor gene product that negatively regulates p21ras and define a "positive" growth role for ras activity in NF1 malignancies.     

The growth factor IL-2 activates p21ras proteins in normal human T lymphocytes.

The T cell growth factor IL-2 induces T cell progression through the cell cycle and ultimately controls T cell mitosis. Here we show that the guanine nucleotide-binding proteins p21ras may be involved in IL-2 signal transduction pathways. IL-2 causes a rapid and prolonged activation of p21ras in both murine and human T cells. The concentration-dependence of IL-2-mediated stimulation of p21ras correlated with IL-2 stimulation of T cell proliferation, which indicates that p21ras activity can be controlled by signals generated via the interaction between IL-2 and its high affinity cellular receptor. These results suggest that p21ras may play a role in the regulation of T cell growth by IL-2.