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PubMed1993

Differential antagonism of Ras biological activity by catalytic and Src homology domains of Ras GTPase activation protein.

Ras p120 GTPase activation protein (GAP), a cytosolic protein, is a negative mediator and potential downstream effector of Ras function. Since membrane association is critical for Ras function, we introduced the Ras membrane-targeting signal (a 19-residue peptide ending in CAAX, where C = cysteine, A = aliphatic amino acid, and X = any amino acid) onto the GAP N-terminal Src homology 2 and 3 and the C-terminal catalytic domains (designated nGAP/CAAX and cGAP/CAAX, respectively) to determine the role of membrane association in GAP function. cGAP/CAAX and full-length GAP/CAAX, but not GAP or nGAP/CAAX, exhibited potent growth inhibitory activity. Whereas both oncogenic and normal Ras activity were inhibited by cGAP/CAAX, nGAP/CAAX, despite lacking the Ras binding domain, inhibited the activity of oncogenic Ras without affecting the action of normal Ras. Altogether, these results demonstrate that membrane association potentiates GAP catalytic activity, support an effector function for GAP, and suggest that normal and oncogenic Ras possess different downstream interactions.

3T3 Cells↗

PubMed1997

A molecular redox switch on p21(ras). Structural basis for the nitric oxide-p21(ras) interaction.

We have identified the site of molecular interaction between nitric oxide (NO) and p21(ras) responsible for initiation of signal transduction. We found that p21(ras) was singly S-nitrosylated and localized this modification to a fragment of p21(ras) containing Cys118. A mutant form of p21(ras), in which Cys118 was changed to a serine residue and termed p21(ras)C118S, was not S-nitrosylated. NO-related species stimulated guanine nucleotide exchange on wild-type p21(ras), resulting in an active form, but not on p21(ras)C118S. Furthermore, in contrast to parental Jurkat T cells, NO-related species did not stimulate mitogen-activated protein kinase activity in cells transfected with p21(ras)C118S. These data indicate that Cys118 is a critical site of redox regulation of p21(ras), and S-nitrosylation of this residue triggers guanine nucleotide exchange and downstream signaling.

Amino Acid Sequence↗

PubMed2001

Inhibition of MAPK activity, cell proliferation, and anchorage-independent growth by N-Ras antisense in an N-ras-transformed human cell line.

Mammalian ras genes encode a family of plasma membrane-bound proteins that function as intermediates in signal transduction pathways involved in cell growth and differentiation. Ras oncogene is frequently involved in neoplastic transformation of different cellular histotypes. In this study, we tested the ability of antisense oligodeoxyribonucleotides (AS-ODN) that have mixed phosphodiester/phosphorothioate backbone, targeted against human N-Ras, to inhibit N-ras gene expression and to specifically interfere with the Ras-dependent activity of mitogen-activated protein kinase (MAPK) in two human cell lines carrying an endogenous N-ras mutated allele at codon 61. Three AS-ODN that inhibit basal MAPK activity have been identified. Moreover, AS-ODN treatment resulted in potent antiproliferative effects in cell culture and great inhibition of N-ras mRNA levels in one of two cell lines. These studies suggest that antisense molecules, targeted against N-Ras, could be of considerable value as a tool to study the N-Ras-specific transduction pathway.

Animals↗

PubMed1997

Induction of the cholesterol metabolic pathway regulates the farnesylation of RAS in embryonic chick heart cells: a new role for ras in regulating the expression of muscarinic receptors and G proteins.

We propose a novel mechanism for the regulation of the processing of Ras and demonstrate a new function for Ras in regulating the expression of cardiac autonomic receptors and their associated G proteins. We have demonstrated previously that induction of endogenous cholesterol synthesis in cultured cardiac myocytes resulted in a coordinated increase in expression of muscarinic receptors, the G protein alpha-subunit, G-alphai2, and the inward rectifying K+ channel, GIRK1. These changes in gene expression were associated with a marked increase in the response of heart cells to parasympathetic stimulation. In this study, we demonstrate that the induction of the cholesterol metabolic pathway regulates Ras processing and that Ras regulates expression of G-alphai2. We show that in primary cultured myocytes most of the RAS is localized to the cytoplasm in an unfarnesylated form. Induction of the cholesterol metabolic pathway results in increased farnesylation and membrane association of RAS. Studies of Ras mutants expressed in cultured heart cells demonstrate that activation of Ras by induction of the cholesterol metabolic pathway results in increased expression of G-alphai2 mRNA. Hence farnesylation of Ras is a regulatable process that plays a novel role in the control of second messenger pathways.

Animals↗

PubMed1995

Human herpesvirus 6A ts suppresses both transformation by H-ras and transcription by the H-ras and human immunodeficiency virus type 1 promoters.

Human herpesvirus 6 strain U1102 (HHV-6A) was shown to contain a 1,473-bp functional transformation suppressor gene (ts). ts exhibited 24% identity and 51% similarity to adeno-associated virus type 2 Rep68/78. Like adeno-associated virus type 2 Rep68/78, HHV-6A ts suppressed H-ras transformation of NIH 3T3 cells. Suppression of H-ras transformation was eliminated by translation termination linker mutation at amino acid 25, 125, or 245. These data indicated the importance of the C-terminal portion of the ts protein. H-ras transformation was suppressed by ts only when H-ras was expressed by its endogenous H-ras promoter and not when it was expressed by the heterologous murine osteosarcoma virus long terminal repeat (LTR). Furthermore, ts suppressed chloramphenicol acetyltransferase (CAT) activity when the CAT gene was expressed from the H-ras promoter but not the murine osteosarcoma virus LTR promoter. Taken together, the data showed that ts suppressed H-ras transformation at the level of the H-ras promoter. To further identify the interaction of ts with transcriptional regulatory elements, the human immunodeficiency virus type 1 (HIV-1) LTR was used. This promoter was selected because it has well-defined transcriptional regulatory elements for both basal and activated transcription, because its activity is inhibited by the Rep68/78 gene, and because both HHV-6 and HIV-1 naturally infect CD4+ T cells in vivo and have been shown to infect the same cell in vitro. ts suppressed expression from both wild-type and upstream mutant HIV-1 LTR-CAT constructs. However, downstream HIV-1 TAR mutations reversed ts suppression, indicating that TAR is one of the critical elements involved. The data presented demonstrated that HHV-6A ts functionally suppressed H-ras transformation and HIV-1 LTR expression and thus that it may be useful in future gene therapy.

3T3 Cells↗

PubMed1997

An intact Raf zinc finger is required for optimal binding to processed Ras and for ras-dependent Raf activation in situ.

The function of the c-Raf-1 zinc finger domain in the activation of the Raf kinase was examined by the creation of variant zinc finger structures. Mutation of Raf Cys 165 and Cys 168 to Ser strongly inhibits the Ras-dependent activation of c-Raf-1 by epidermal growth factor (EGF). Deletion of the Raf zinc finger and replacement with a homologous zinc finger from protein kinase C gamma (PKC gamma) (to give gamma/Raf) also abrogates EGF-induced activation but enables a vigorous phorbol myristate acetate (PMA)-induced activation. PMA activation of gamma/Raf does not require endogenous Ras or PKCs and probably occurs through a PMA-induced recruitment of gamma/Raf to the plasma membrane. The impaired ability of EGF to activate the Raf zinc finger variants in situ is attributable, at least in part, to a major decrement in their binding to Ras-GTP; both Raf zinc finger variants exhibit decreased association with Ras (V12) in situ upon coexpression in COS cells, as well as diminished binding in vitro to immobilized, processed COS recombinant Ras(V12)-GTP. In contrast, Raf binding to unprocessed COS or prokaryotic recombinant Ras-GTP is unaffected by Raf zinc finger mutation. Thus, the Raf zinc finger contributes an important component to the overall binding to Ras-GTP in situ, through an interaction between the zinc finger and an epitope on Ras, distinct from the effector loop, that is present only on prenylated Ras.

Amino Acid Sequence↗

PubMed2003

A novel Ras inhibitor, Eri1, engages yeast Ras at the endoplasmic reticulum.

Ras oncoproteins are monomeric GTPases that link signals from the cell surface to pathways that regulate cell proliferation and differentiation. Constitutively active mutant forms of Ras are found in ca. 30% of human tumors. Here we report the isolation of a novel gene from Saccharomyces cerevisiae, designated ERI1 (for endoplasmic reticulum-associated Ras inhibitor 1), which behaves genetically as an inhibitor of Ras signaling. ERI1 encodes a 68-amino-acid protein that associates in vivo with GTP-bound Ras in a manner that requires an intact Ras-effector loop, suggesting that Eri1 competes for the same binding site as Ras target proteins. We show that Eri1 localizes primarily to the membrane of the endoplasmic reticulum (ER), where it engages Ras. The recent demonstration that signaling from mammalian Ras is not restricted to the cell surface but can also proceed from the cytoplasmic face of the ER suggests a regulatory function for Eri1 at that membrane.

Amino Acid Sequence↗

PubMed1988

Requirement of one functional RAS gene and inability of an oncogenic ras variant to mediate the glucose-induced cyclic AMP signal in the yeast Saccharomyces cerevisiae.

Addition of glucose to Saccharomyces cerevisiae cells grown on a nonfermentable carbon source triggers a cyclic AMP (cAMP) signal, which induces a protein phosphorylation cascade. In a yeast strain lacking functional RAS1 and RAS2 genes and containing a bcy mutation to suppress the lethality of RAS deficiency, the cAMP signal was absent. Addition of dinitrophenol, which stimulates in vivo cAMP synthesis by lowering intracellular pH, also did not enhance the cAMP level. A bcy control strain, with functional RAS genes present, showed cAMP responses similar to those of a wild-type strain. In disruption mutants containing either a functional RAS1 gene or a functional RAS2 gene, the cAMP signal was not significantly different from the one in wild-type cells, indicating that RAS function cannot be a limiting factor for cAMP synthesis during induction of the signal. Compared with wild-type cells, the cAMP signal decreased in intensity with increasing temperature in a ras2 disruption mutant. When the mutant RAS2Val-19, which carries the equivalent of the human H-rasVal-12 oncogene, was grown under conditions in which RAS1 expression is repressed, the cAMP signal was absent. The oncogene product is known to be deficient in GTPase activity. However, the amino acid change at position 19 (or 12 in the corresponding human oncogene product) might also have other effects, such as abolishing receptor interaction. Such an additional effect probably provides a better explanation for the lack of signal transmission than the impaired GTPase activity. When the RAS2Val-19 mutant was grown under conditions in which RAS1 is expressed, the cAMP signal was present but significantly delayed compared with the signal in wild-type cells. This indicates that oncogenic RAS proteins inhibit normal functioning of wild-type RAS proteins in vivo and also that in spite of the presence of the RAS2(Val-19) oncogene, adenyl cyclase is not maximally stimulated in vivo. Expression of only the RAS(Val-19) gene product also prevented most of the stimulation of cAMP synthesis by dinitrophenol, indicating that lowered intracellular pH does not act directly on adenyl cyclase but on a step earlier in the activation pathway of the enzyme. The results obtained with the control bcy strain, the RAS2(Val-19) strain under conditions in which RAS1 is expressed, and with dinitrophenol show that the inability of the oncogene product to mediate the cAMP signal is not due to feedback inhibition by the high protein kinase activity in strains containing the RAS2(Val-19) oncogene. Hence, the present results show that the RAS protein in S. cerevisiae are involved in the transmission of the glucose-induced cAMP signal and that the oncogenic RAS protein is unable to act as a signal transducer. The RAS protein in S. cerevisiae apparently act similarly to the Gs proteins of mammalian adenyl cyclase, but instead of being involved in hormone signal transmission, they function in a nutrient-induced signal transmission pathway.

Cyclic AMP↗

PubMed2000

Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras.

We have examined the functional consequences of ADP-ribosyltransferase modification of Ras by the exoenzyme S (ExoS) protein of Pseudomonas aeruginosa. ExoS has been shown previously to ADP-ribosylate a number of proteins, including members of the Ras superfamily, which play an essential role in the processes of cell proliferation, differentiation, motility and cell division. HeLa and NIH3T3 cells were infected with ExoS protein, which was delivered via the type III secretion system of the heterologous host Yersinia pseudotuberculosis. Infection of mammalian cells with ExoS results in a change in the ratio of GTP/GDP bound directly to Ras in vivo. This ADP-ribosylation of Ras in vivo is mediated by the C-terminal domain of ExoS. Further, ExoS ADP-ribosylation of Ras in vivo inhibits activation of Ras and the ability to interact with the Ras binding domain of Raf upon stimulation with epidermal growth factor (EGF). In the present study, we show that ExoS activity does not interfere with EGF receptor phosphorylation itself, nor with the formation of a Grb2-activated Shc complex upon EGF stimulation, consistent with ExoS blockage of this mitogenic signalling pathway at the level of Ras. This is further supported by our observation of a substantial inhibition of extracellular signal-regulated kinase and protein kinase B/Akt kinase activation in response to EGF upon ExoS infection. In conclusion, in the present study, the consequences of ExoS infection on Ras effector pathway in vivo have been defined.

3T3 Cells↗

PubMed1991

Organization of the human N-ras locus: characterization of a gene located immediately upstream of N-ras.

The mammalian ras genes have been implicated in a wide variety of natural and experimental tumors. They code for small GTP binding proteins which are believed to play a central role in the control of cellular proliferation and differentiation. We have investigated the transcriptional organization of the human N-ras locus and characterized a transcription unit located immediately upstream of N-ras, which we designate by NRU for N-ras Upstream. NRU messages contain an open reading frame of 767 amino acids which shows no similarity with the ras proteins and provides no clue to the function of the corresponding protein. Of the order of 150 nucleotides separate the N-ras transcription initiation sites from the last NRU polyadenylation site and the same organization is present in the murine genome. Both genes are simultaneously expressed in all the cell lines and murine tissues we have analysed, and in all cases NRU messages accumulate to a higher level than those of N-ras. The small intergenic distance implies that, during transcription of NRU, RNA polymerases transcribe a substantial part of the N-ras gene, suggesting that this genetic organization participates in the regulation of N-ras expression.

Animals↗

PubMed1989

ras mutations in human melanotic lesions: K-ras activation is a frequent and early event in melanoma development.

We have used polymerase chain reaction (PCR), an amplification procedure, and oligonucleotide hybridization to detect ras gene point mutations in DNA from melanoma tumor samples. Genomic DNA was examined from 40 specimens of melanotic lesions, including benign nevi, primary melanomas, lymph node metastases, and systemic metastases. Adjacent normal skin or peripheral blood was analyzed as control material in 28 cases. ras mutations were detected overall in 25% of malignant tumors. In addition, mutations of all three ras genes were detected. We observed ras mutations in 2 of 4 benign atypical nevi (2 X K12), 4 of 22 primary melanomas (3 X K12, 1 X H12, 1 X N61), and 4 of 14 secondary (5 X K12, 1 X N61) tumors. One with a primary melanoma had concurrent K12 and H12, and two patients with secondary tumors had concurrent K12/N61 and K12 Asp/K12 Val mutations, respectively, making a total of 10 of 40 (25%) patients with ras mutations. This is the first demonstration of K-ras mutations in human melanoma. The presence of K-ras mutations in nevi, putative melanoma precursors, suggests that ras activation may be an early event in melanoma development. No correlation between tumor thickness and the presence of a ras mutation was observed.

Adult↗

PubMed1988

Mutation of Ki-ras and N-ras oncogenes in myelodysplastic syndromes.

Somatic mutation of the N-ras oncogene occurs frequently in de novo acute myeloid leukemia (AML). By virtue of their relation to AML, myelodysplastic syndromes (MDS) provide an in vivo model of human leukemogenesis. By using a strategy for analysis of gene mutation based on in vitro amplification of target sequences by the polymerase chain reaction (PCR) and selective oligonucleotide hybridization we analyzed the mutational status of codons 12, 13, and 61 of Ha-ras, K-ras, and N-ras in peripheral blood (PB) and/or bone marrow (BM) in 34 cases of primary MDS. Mutations at codon 12 of Ki-ras or N-ras were detected in three cases (9%): one of six cases of refractory anemia with excess blasts (RAEB) and two of nine cases of chronic myelomonocytic leukemia (CMML). The nucleotide substitution differed in each. In all cases the mutant allele was detectable in PB cells. A sustained hematologic remission was achieved after low-dose cytarabine therapy in the case of RAEB. Neither case of CMML exhibited signs of disease progression during follow-up at 7 and 12 months. In contrast, four of 31 patients without the ras mutation underwent transformation to AML within 12 months of genetic analysis. We conclude that ras mutations in MDS are heterogeneous and may develop at an early stage during the evolution of MDS. Their detection in PB cells illustrates the potential utility of ras mutation as a clonal marker in myeloid malignancy.

Genes, ras↗

PubMed1995

[Amplification of proto-oncogenes C-myc, C-N-ras, C-Ki-ras, C-erbB2 in ovarian carcinoma].

OBJECTIVE: To observe the amplification rate of C-myc, C-N-ras, C-Ki-ras, C-erbB2 in human ovarian carcinoma. METHODS: Southern blot hybridization of DNA was employed, pathological diagnosis was made from fresh tissues. The beta-globin gene was used as an internal control. RESULTS: The amplification rate of C-myc, C-N-ras, C-Ki-ras and C-erbB2 in ovarian carcinoma was 50%, 44%, 31% and 25% respectively. The amplification rates of C-myc, C-erbB2 in stage III and IV were all significantly greater than that in stage I (P < 0.01). The amplification rate of C-N-ras in stage I was also significantly greater than that in stage III (P < 0.01). The amplification rate of C-Ki-ras in stage I was significantly greater than that in stage III or IV (P < 0.01). CONCLUSIONS: The amplification of C-Ki-ras and C-N-ras took place chiefly in cases of the early stages and in cases with good differentiation. The amplification of C-N-ras was also found in cases of advanced stages. The amplifications of C-myc and C-erbB2 were chiefly found in cases above stage III and in cases with poor differentiation, 83% of the patients who died were found to have amplifications of more than 2 proto-oncogenes, with the amplification of C-erbB2 involved in all of them.

Blotting, Southern↗

PubMed1994

Transgenic mice provide genetic evidence that transforming growth factor alpha promotes skin tumorigenesis via H-ras-dependent and H-ras-independent pathways.

The epidermal growth factor receptor (EGFR), which mediates the mitogenic activity of transforming growth factor alpha (TGF-alpha), has been shown to activate Ras in cultured cells through well-defined intermediary proteins. To examine the in vivo relationship between EGFR and Ras, chemical carcinogenesis of TGF-alpha transgenic mouse skin was chosen as an experimental model. Transgenic mice overexpressing TGF-alpha in a wide variety of epithelial tissues by virtue of a metallothionein promoter demonstrate a multitude of premalignant and neoplastic lesions but not spontaneous skin tumors. Transgenic skin was initiated with a single dose of 7,12-dimethylbenz[a]anthracene (DMBA), shown previously to induce, in concert with a tumor promoter, murine papillomas that consistently contain specific H-ras mutations. Virtually all DMBA-treated TGF-alpha transgenic mice, but not treated control animals, developed hyperplasias, papillomas, sebaceous adenomas, and more infrequently, sebaceous and squamous cell carcinomas. Therefore, TGF-alpha functions as an autonomous tumor promoter in DMBA-initiated transgenic skin. Skin tumors could be separated into two mutually exclusive genetic classes. In tumors harboring mutant H-ras, TGF-alpha transgene expression was relatively low and essentially unchanged relative to untreated skin; however, only 42% of skin tumors contained mutations in H-ras. Conversely, in most tumors with wild-type H-ras, transgenic TGF-alpha transcripts were enhanced 10- to 20-fold. These results suggest that strong constitutive EGFR stimulation, through TGF-alpha transgene overexpression, can substitute functionally for mutational activation of H-ras in skin tumorigenesis. Moreover, because H-ras mutational activation could not induce skin tumors without TGF-alpha transgene activity, simultaneous stimulation of an EGFR-mediated H-Ras-independent pathway appears to be required for tumor development as well.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1997

K-ras gene mutations in normal colorectal tissues from K-ras mutation-positive colorectal cancer patients.

K-ras gene mutations have been reported as early events in colorectal tumorigenesis, but their role in tumor initiation and development is still unclear. To analyze and compare K-ras mutational patterns between colorectal tissues at different stages of tumor progression in individual patients, 65 colorectal tissue samples, including carcinoma, adenoma, histologically normal mucosa, submucosal muscularis propria, and histologically normal mucosa distant from tumor, were obtained from 13 patients with colorectal cancer. In addition, normal mucosal tissues obtained from four normal individuals were analyzed. Each of the 13 tumors was shown previously to harbor a mutation in either codon 12 or 13 of the K-ras gene by direct sequencing. These tissues were reanalyzed, using the recently established mutant allele enrichment + denaturing gradient gel electrophoresis method, which can detect one mutant allele in 10(4)-10(5) normal alleles, thus allowing for the analysis of infrequent cells bearing mutations against the background of wild-type cells. No K-ras codon 12 mutation was detected by this method in the histologically normal mucosal tissues sampled at the margin of resection distant from the tumor or in those obtained from four normal individuals. On the other hand, these mutations were detected in 9 of 10 adenoma and 6 of 10 mucosa samples from 10 patients with known K-ras codon 12 mutations, and also in 2 of 3 carcinoma, 2 of 3 adenoma, and 1 of 3 mucosa samples obtained from 3 patients with known K-ras codon 13 mutations. Thus, K-ras codon 12 mutations were found to occur with a high frequency (53.8%) in histologically normal mucosa adjacent to tumors of patients with K-ras mutation-positive colorectal cancer, suggesting that they may be useful biomarkers for early detection of colorectal cancer. Furthermore, multiple K-ras mutations were found in tissues of nearly half of the 13 patients, indicating that distinct evolutionary subclones may be involved in the development of tumor in some patients with colorectal cancer.

Adenoma↗

PubMed2007

Mechanisms of guanosine triphosphate hydrolysis by Ras and Ras-GAP proteins as rationalized by ab initio QM/MM simulations.

The hydrolysis reaction of guanosine triphosphate (GTP) by p21(ras) (Ras) has been modeled by using the ab initio type quantum mechanical-molecular mechanical simulations. Initial geometry configurations have been prompted by atomic coordinates of the crystal structure (PDBID: 1QRA) corresponding to the prehydrolysis state of Ras in complex with GTP. Multiple searches of minimum energy geometry configurations consistent with the hydrogen bond networks have been performed, resulting in a series of stationary points on the potential energy surface for reaction intermediates and transition states. It is shown that the minimum energy reaction path is consistent with an assumption of a two-step mechanism of GTP hydrolysis. At the first stage, a unified action of the nearest residues of Ras and the nearest water molecules results in a substantial spatial separation of the gamma-phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low barrier (16.7 kcal/mol) transition state TS1. At the second stage, the inorganic phosphate is formed in consequence of proton transfers mediated by two water molecules and assisted by the Gln61 residue from Ras. The highest transition state at this segment, TS3, is estimated to have an energy 7.5 kcal/mol above the enzyme-substrate complex. The results of simulations are compared to the previous findings for the GTP hydrolysis in the Ras-GAP (p21(ras)-p120(GAP)) protein complex. Conclusions of the modeling lead to a better understanding of the anticatalytic effect of cancer causing mutation of Gln61 from Ras, which has been debated in recent years.

Amino Acid Sequence↗

PubMedDate not supplied

The exchange factor Ras-GRF2 activates Ras-dependent and Rac-dependent mitogen-activated protein kinase pathways.

Ras and Rac are membrane-associated GTPases that function as molecular switches activating intracellular mitogen-activated protein kinase (MAPK) cascades and other effector pathways in response to extracellular signals [1]. Activation of Ras and Rac into their GTP-bound conformations is directly controlled by specific guanine-nucleotide exchange factors (GEFs), which catalyze GDP release. Several Ras-specific GEFs that are related to the budding yeast protein Cdc25p have been described, whereas GEFs for Rac-related GTPases contain a region that is homologous to the oncoprotein DbI [2-3]. The Ras-GRF1 and Ras-GRF2 proteins, which couple Ras activation to serpentine receptors and calcium signals, contain both Cdc25 and DbI homology (DH) regions [3-4]. Here, we demonstrate that Ras-GRF2 is a bifunctional signaling protein that is able to bind and activate Ras and Rac, and thereby coordinate the activation of the extracellular-signal-regulated kinase (ERK) and stress-activated protein kinase (SAPK) pathways.

Anisomycin↗

PubMed2000

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts.

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25Mm-GEF), conserved in all ras-specific GEFs identified so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to efficiently displace wild-type GEF from p21ras and to originate a stable ras/GEF binary complex due to the reduced affinity of the nucleotide-free ras/GEF complex for the incoming nucleotide. We show here that this 'ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse fibroblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEFW1056E in stable transfected cells strongly reduces intracellular ras-GTP levels in k-ras transformed fibroblasts. Accordingly, the transfected fibroblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-specific GEFs as a tool to down-regulate tumor growth is discussed.

Animals↗