scholarly journals Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor.

1993 ◽  
Vol 13 (12) ◽  
pp. 7718-7724 ◽  
Author(s):  
H Cen ◽  
A G Papageorge ◽  
W C Vass ◽  
K E Zhang ◽  
D R Lowy
Keyword(s):  
The Other ◽  
Guanine Nucleotide ◽  
3T3 Cells ◽  
Ras Protein ◽  
Exchange Factor ◽  
Type Iv ◽  
Serum Dependent ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
In Cells

Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state. We have recently identified four types (I to IV) of apparently full-length cDNAs from a single mammalian gene, called CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S. cerevisiae. The largest cDNA (type IV) is brain specific, with the other three classes, although they have distinct 5' ends, essentially representing progressive N-terminal deletions of this cDNA. When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras. Serum stimulation of these transformants lead to a further increase in GTP.Ras only in cells expressing the type IV cDNA. Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimulate guanine nucleotide release from GDP.Ras in vitro. When NIH 3T3 cells and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras protein and the other encoding a cytosolic Ras protein, the basal levels of GTP bound to both forms of the mutant Ras protein were significantly higher in the cells expressing the type IV protein. However, serum increased the level of GTP bound to the membrane-associated mutant Ras protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein. We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP.Ras, and the serum-dependent increase in GTP.Ras by exogenous CDC25Mm or by endogenous exchange factors probably requires membrane association of both Ras and the exchange factor.

Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor

1993 ◽  
Vol 13 (12) ◽  
pp. 7718-7724
Author(s):  
H Cen ◽  
A G Papageorge ◽  
W C Vass ◽  
K E Zhang ◽  
D R Lowy
Keyword(s):  
The Other ◽  
Guanine Nucleotide ◽  
3T3 Cells ◽  
Ras Protein ◽  
Exchange Factor ◽  
Type Iv ◽  
Serum Dependent ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
In Cells

Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state. We have recently identified four types (I to IV) of apparently full-length cDNAs from a single mammalian gene, called CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S. cerevisiae. The largest cDNA (type IV) is brain specific, with the other three classes, although they have distinct 5' ends, essentially representing progressive N-terminal deletions of this cDNA. When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras. Serum stimulation of these transformants lead to a further increase in GTP.Ras only in cells expressing the type IV cDNA. Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimulate guanine nucleotide release from GDP.Ras in vitro. When NIH 3T3 cells and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras protein and the other encoding a cytosolic Ras protein, the basal levels of GTP bound to both forms of the mutant Ras protein were significantly higher in the cells expressing the type IV protein. However, serum increased the level of GTP bound to the membrane-associated mutant Ras protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein. We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP.Ras, and the serum-dependent increase in GTP.Ras by exogenous CDC25Mm or by endogenous exchange factors probably requires membrane association of both Ras and the exchange factor.

scholarly journals Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein.

1994 ◽  
Vol 14 (2) ◽  
pp. 906-913 ◽  
Author(s):  
E Gulbins ◽  
K M Coggeshall ◽  
C Langlet ◽  
G Baier ◽  
N Bonnefoy-Berard ◽  
...  
Keyword(s):  
Map Kinases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
3T3 Cells ◽  
Transfected Cells ◽  
Guanine Nucleotide Exchange ◽  
Nih 3T3 ◽  
Exchange Activity ◽  
Nih 3T3 Cells

We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role of Vav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56lck displayed GEF activity against Ras but not against recombinant RacI, RacII, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a approximately 10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras. GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras. GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation.

scholarly journals The Ras Mutant D119N Is Both Dominant Negative and Activated

1999 ◽  
Vol 19 (9) ◽  
pp. 6297-6305 ◽  
Author(s):  
Robbert H. Cool ◽  
Gudula Schmidt ◽  
Christian U. Lenzen ◽  
Heino Prinz ◽  
Dorothee Vogt ◽  
...  
Keyword(s):  
Calf Serum ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Binding Motif ◽  
Loss Of Function ◽  
3T3 Cells ◽  
Effector Molecules ◽  
Exchange Factor ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT The introduction of mutation D119N (or its homolog) in the NKxD nucleotide binding motif of various Ras-like proteins produces constitutively activated or dominant-negative effects, depending on the system and assay. Here we show that Ras(D119N) has an inhibitory effect at a cell-specific concentration in PC12 and NIH 3T3 cells. Biochemical data strongly suggest that the predominant effect of mutation D119N in Ras—a strong decrease in nucleotide affinity—enables this mutant (i) to sequester its guanine nucleotide exchange factor, as well as (ii) to rapidly bind GTP, independent of the regulatory action of the exchange factor. Since mutation D119N does not affect the interaction between Ras and effector molecules, the latter effect causes Ras(D119N) to act as an activated Ras protein at concentrations higher than that of the exchange factor. In comparison, Ras(S17N), which also shows a strongly decreased nucleotide affinity, does not bind to effector molecules. These results point to two important prerequisites of dominant-negative Ras mutants: an increased relative affinity of the mutated Ras for the exchange factor over that for the nucleotide and an inability to interact with the effector or effectors. Remarkably, the introduction of a second, partial-loss-of-function, mutation turns Ras(D119N) into a strong dominant-negative mutant even at high concentrations, as demonstrated by the inhibitory effects of Ras(E37G/D119N) on nerve growth factor-mediated neurite outgrowth in PC12 cells and Ras(T35S/D119N) on fetal calf serum-mediated DNA synthesis in NIH 3T3 cells. Interpretations of these results are discussed.

Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates

1988 ◽  
Vol 179 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Shigemi Kato ◽  
Theresa L. Ben ◽  
Luigi M. De Luca
Keyword(s):  
Type Iv Collagen ◽  
Phorbol Esters ◽  
3T3 Cells ◽  
Type Iv ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Desensitization of the Ca2+-mobilizing system to serum growth factors by Ha-ras and v-mos

1988 ◽  
Vol 8 (10) ◽  
pp. 4212-4216
Author(s):  
K Maly ◽  
W Doppler ◽  
H Oberhuber ◽  
H Meusburger ◽  
J Hofmann ◽  
...  
Keyword(s):  
Growth Factors ◽  
Intracellular Ph ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
3T3 Cells ◽  
Ras Gene ◽  
Phosphatidylinositol Turnover ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
In Cells

An elevation of the intracellular pH and a rise in the cytoplasmic Ca2+ concentration are considered important mitogenic signals which are observed after stimulation by various growth factors. In a preceding report it was demonstrated that the expression of Ha-ras or v-mos in cells transfected with Ha-ras or v-mos, respectively, leads to an activation of the Na+/H+ antiporter and a concomitant rise in intracellular pH (W. Doppler, R. Jaggi, and B. Groner, Gene 54:145-151, 1987). This report describes the effect of the Ha-ras and v-mos oncogenes on intracellular Ca2+ release. The expression of Ha-ras in NIH 3T3 cells carrying a glucocorticoid-inducible transforming Ha-ras gene caused a desensitization of the Ca2+-mobilizing system to serum growth factors. The induction of p21ras in cells carrying the corresponding glucocorticoid-inducible proto-oncogene did not affect the Ca2+ response to growth factors. Conditions leading to the expression of the transforming Ha-ras gene but not those causing the induction of the normal Ha-ras gene yielded an increase in phosphatidylinositol turnover and a concomitant rise in inositol phosphates. Results similar to those obtained with the transforming Ha-ras gene were seen after the expression of v-mos. The data are consistent with a mechanism in which expression of the transforming Ha-ras gene leads to a release of Ca2+ from intracellular stores via elevated levels of inositol trisphosphate.

Thiophosphate and selenite conversely modulate cell death induced by glutathione depletion or cisplatin: effects related to activity and Sec contents of thioredoxin reductase

2012 ◽  
Vol 447 (1) ◽  
pp. 167-174 ◽  
Author(s):  
Xiaoxiao Peng ◽  
Jianqiang Xu ◽  
Elias S. J. Arnér
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Thioredoxin Reductase ◽  
Glutathione Depletion ◽  
3T3 Cells ◽  
Drug Induced ◽  
Direct Cytotoxicity ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
In Cells

Thiophosphate (SPO3) was recently shown to promote cysteine insertion at Sec (selenocysteine)-encoding UGA codons during selenoprotein synthesis. We reported previously that irreversible targeting by cDDP [cis-diamminedichloroplatinum(II) or cisplatin] of the Sec residue in TrxR1 (thioredoxin reductase 1) contributes to cDDP cytotoxicity. This effect could possibly be attenuated in cells expressing less reactive Sec-to-cysteine-substituted TrxR1 variants, or pronounced in cells with higher levels of Sec-containing TrxR1. To test this, we supplemented cells with either SPO3 or selenium and subsequently determined total as well as specific activities of cellular TrxR1, together with extent of drug-induced cell death. We found that cDDP became less cytotoxic after incubation of A549 or HCT116 cells with lower SPO3 concentrations (100–300 μM), whereas higher SPO3 (>300 μM) had pronounced direct cytotoxicity. NIH 3T3 cells showed low basal TrxR1 activity and high susceptibility to SPO3 cytotoxicity, or to glutathione depletion. Supplementing NIH 3T3 cells with selenite, however, gave increased cellular TrxR1 activity with concomitantly decreased dependence on glutathione, whereas the susceptibility to cDDP increased. The results suggest molecular mechanisms by which the selenium status of cells can affect their glutathione dependence while modulating the cytotoxicity of drugs that target TrxR1.

scholarly journals Desensitization of the Ca2+-mobilizing system to serum growth factors by Ha-ras and v-mos.

1988 ◽  
Vol 8 (10) ◽  
pp. 4212-4216 ◽  
Author(s):  
K Maly ◽  
W Doppler ◽  
H Oberhuber ◽  
H Meusburger ◽  
J Hofmann ◽  
...  
Keyword(s):  
Growth Factors ◽  
Intracellular Ph ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
3T3 Cells ◽  
Ras Gene ◽  
Phosphatidylinositol Turnover ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
In Cells

An elevation of the intracellular pH and a rise in the cytoplasmic Ca2+ concentration are considered important mitogenic signals which are observed after stimulation by various growth factors. In a preceding report it was demonstrated that the expression of Ha-ras or v-mos in cells transfected with Ha-ras or v-mos, respectively, leads to an activation of the Na+/H+ antiporter and a concomitant rise in intracellular pH (W. Doppler, R. Jaggi, and B. Groner, Gene 54:145-151, 1987). This report describes the effect of the Ha-ras and v-mos oncogenes on intracellular Ca2+ release. The expression of Ha-ras in NIH 3T3 cells carrying a glucocorticoid-inducible transforming Ha-ras gene caused a desensitization of the Ca2+-mobilizing system to serum growth factors. The induction of p21ras in cells carrying the corresponding glucocorticoid-inducible proto-oncogene did not affect the Ca2+ response to growth factors. Conditions leading to the expression of the transforming Ha-ras gene but not those causing the induction of the normal Ha-ras gene yielded an increase in phosphatidylinositol turnover and a concomitant rise in inositol phosphates. Results similar to those obtained with the transforming Ha-ras gene were seen after the expression of v-mos. The data are consistent with a mechanism in which expression of the transforming Ha-ras gene leads to a release of Ca2+ from intracellular stores via elevated levels of inositol trisphosphate.

scholarly journals Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

1988 ◽  
Vol 8 (8) ◽  
pp. 3235-3243 ◽  
Author(s):  
L A Feig ◽  
G M Cooper
Keyword(s):  
Cell Proliferation ◽  
Inhibitory Effect ◽  
3T3 Cells ◽  
Ras Gene ◽  
Ras Protein ◽  
Mammalian Expression ◽  
Guanine Nucleotide Binding ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.

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