Effect of Ha-ras on phosphatidylinositol metabolism, Na+/H+-antiporter and mobilization of intracellular calcium (original) (raw)
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Proceedings of the National Academy of Sciences, 1987
Data indicating that the 21-kDa protein (p21) Harvey-ras gene product shares sequence homology with guanine nucleotide-binding proteins (G proteins) has stimulated research on the influence(s) of p21 on G-protein-regulated systems in vertebrate cells. Our previous work demonstrated that NIH-3T3 mouse cells expressing high levels of the cellular ras oncogene isolated from the EJ human bladder carcinoma (EJ-ras) exhibited reduced hormone-stimulated adenylate cyclase activity. We now report that in these cells another enzyme system thought to be regulated by G proteins is inhibited, namely phospholipases A2 and C. NIH-3T3 cells incubated in plasma-derived serum release significant levels of prostaglandin E2 (PGE2) as determined by radioimmunoassay when exposed to platelet-derived growth factor (PDGF) at 2 units/ ml; the levels of PGE2 released from EJ-ras-transfected cells are only 3% those of controls despite a similar basal (unstimulated) release from control and EJ-ras-transfected cells. The lack of PDGF-stimulated PGE2 release from EJ-ras-transfected cells is not due to a defect in the prostaglandin cyclooxygenase enzyme, since incubation of control cells and EJ-ras-transfected tells in 0.33, 3.3, or 33 IAM arachidonate resulted in identical levels of PGE2 release. The lack of PDGF-stimulated PGE2 release from EJ-ras-transfected cells also does not result from the loss of functional PDGF receptors. EJ-ras-transformed cells bind 70% as much '25I-labeled PDGF as control cells and are stimulated to incorporate [3H]thymidine and to proliferate after exposure to PDGF. Moreover, this inhibition is not likely the result of a secondary cellular effect related to the transformed phenotype, since NIH-3T3 cells transformed by v-src released PGE2 at wild-type levels after exposure to PDGF. Determination of total water-soluble inositolphospholipids and changes in the specific activities of phosphatidylcholine in control and EJ-ras-transfected cells demonstrated that PDGF-stimulated phospholipase C and A2 activities are inhibited in the EJ-ras-transfected cells.
Molecular and cellular biology, 1991
A set of genes is rapidly inducible when quiescent fibroblasts are stimulated by growth factors or by the activation of temperature-sensitive retroviral protein-tyrosine kinases. Most of these so-called immediate-early genes were cloned by differential cDNA hybridization. DNA sequence analysis identified many of them as putative members of the growth factor or of the transcription factor gene family, suggesting a role in signal transmission during the G0-to-G1 transition. In this study, we identified one of the genes that are rapidly inducible by the retroviral protein-tyrosine kinases v-Src and v-Fps of Rous sarcoma virus and Fujinami sarcoma virus, respectively, as the rhoB gene, a member of the ras gene superfamily whose products are GTP-binding proteins, rhoB is transiently activated at the transcriptional level by v-Fps and by epidermal growth factor. Its labile RNA is inducible in the presence of cycloheximide but not of actinomycin D. rhoB is strongly induced by epidermal gro...
Desensitization of the Ca2+-mobilizing system to serum growth factors by Ha-ras and v-mos
Molecular and cellular biology, 1988
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 tho...
1997
Glycerophosphoinositols are phosphoinositide metabolites whose levels are constitutively elevated in Ras-transformed cells. Here, we show that one of these compounds, glycerophosphoinositol-4-phosphate (GroPlns-4-P) responds acutely to the stimulation of the epidermal growth factor receptor, with a fast, massive and transient increase. The mechanism leading to GroPIns-4-P formation involves the activation of phosphoinositide-3 kinase and the small GTP-binding protein Rac, since GroPIns-4-P was neither formed in cells expressing the dominant negative form of Rac nor in cells treated with the phosphoinositide-3 kinase inhibitor wortmannin. GroPIns-4-P has been previously shown to inhibit adenylyl cyclase. Accordingly, epidermal growth factor also decreased the basal, cholera toxin-stimulated, and forskolin-stimulated cyclic AMP levels with kinetics similar to those of GroPIns4-P formation, suggesting that GroPIns-4-P mediates this inhibitory effect. The hormone-induced formation of GroPIns-4-P was detected in several cell lines of various origin, suggesting that GroPIns4-P is a novel intracellular messenger of the Ras pathway, possibly able to convey information from tyrosine kinase receptors to the cyclic AMP cascade.
Association of p21ras with phosphatidylinositol 3-kinase
Proceedings of the National Academy of Sciences, 1991
In mammalian cells, ras genes code for 21-kDa GTP-binding proteins. Increased expression and mutations in specific amino acids have been closely linked to alterations of normal cell morphology, growth, and differentiation and, in particular, to neoplastic transformation. The signal transduction induced by these p21' proteins is largely unknown; however, the signaling pathways of several growth factors have been reported to involve phosphatidylinositol (Ptdlns) 3-kinase. In the present study of a Ha-ras-transformed epithelial cell line, we demonstrated increased Ptdlns 3-kinase activity in anti-phosphotyrosine and anti-receptor (insulin and hybrid insulin-like growth factor I) immunoprecipitates of cells that had been stimulated with insulin or insulin-like growth factor I. The PtdIns 3-kinase activity was also immunoprecipitated in these experiments by the anti-Ras monoclonal antibody Y13-259. The specificity ofthis association with p21t was ascertained by the neutralizing effect of the antigen peptide and the absence of PtdIns 3-kinase activity in Y13-259 immunoprecipitates from cells in which the ras gene was turned off.
Platelet-derived growth factor does not induce c-fos in NIH 3T3 cells expressing the EJ-ras oncogene
Molecular and Cellular Biology, 1988
Platelet-derived growth factor (PDGF), the calcium ionophore A23187, and the tumor promoter phorbol myristate acetate stimulated c-fos mRNA levels in control NIH 3T3 cells. However, NIH 3T3 cells transformed by EJ-ras DNA transfection, which have diminished PDGF-stimulated phospholipase C activity, showed a 95% reduction in PDGF-stimulated c-fos mRNA levels. The responses to A23187 and phorbol myristate acetate were also attenuated, but not as severely as the PDGF-mediated induction. The reduction in PDGF-stimulated c-fos induction did not appear to be a general result of cellular transformation, since src-transformed NIH 3T3 cells displayed a strong PDGF-stimulated c-fos induction. Despite the reduction in PDGF-stimulated c-fos induction, EJ-ras-transformed cells still responded mitogenically to PDGF. These data suggest that the magnitude of c-fos induction cannot be directly correlated with PDGF-stimulated mitogenesis in EJ-ras-transformed NIH 3T3 cells.
FEBS Letters, 1993
Having determined the complete amino acid sequence of a cytosolic phosphatase purified from bovine liver, we studied the role of this enzyme (referred to as 'PTPase') in the control of cell proliferation. We used NIH/3T3 fibroblasts, both normal and transformed by the oncogenes v-erbB, v-src, and v-raf: a synthetic gene coding for PTPase was transfected into, and overexpressed in, normal and transformed NIH/3T3 cells with resulting inhibition of cell growth. Inhibition of proliferation correlated with the level of foreign PTPase; growth in soft agar was also inhibited in transformants overexpressing the enzyme. However, PTPase overexpression did not inhibit the rapid turnover of inositol lipids stimulated by platelet-derived growth factor. We conclude that this novel PTPase is active on cell type-specific signalling substrates that control normal and transformed fibroblast proliferation.