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1
Naz, R. K., K. Ahmad, and P. Kaplan. "Expression and function of ras proto-oncogene proteins in human sperm cells." Journal of Cell Science 102, no. 3 (July 1, 1992): 487–94. http://dx.doi.org/10.1242/jcs.102.3.487.
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The presence and role of c-ras proteins were investigated in mature human sperm cells. The v-H-ras monoclonal antibody (mAb) against the c-ras protein, p21, reacted specifically with the acrosomal region of methanol-fixed as well as unfixed-live capacitated and non-capacitated human sperm cell in the indirect immunofluorescence technique. The v-H-ras mAb predominantly recognized c-ras protein of 21 kDa on the Western blot of lithium diiodosalicylate (LIS)-solubilized human sperm preparation. The incubation of sperm cells with v-H-ras mAb affected the sperm cell function in the human sperm penetration assay. The antibody significantly reduced the acrosome reaction and release of acrosin activity from the sperm cells. There was no effect of the mAb on percentage motility, although the mAb significantly affected various motility characteristics such as linearity, amplitude of lateral head displacement (ALH) and beat frequency, the motility parameters involved in the hyperactivation phenomenon of sperm cells leading to capacitation and acrosome reaction. These results suggest that the c-ras or c-ras-like proteins are present in mature sperm cell and may have a role in capacitation and/or acrosome reaction of human sperm cell.
2
Swanson, M. E., A. M. Elste, S. M. Greenberg, J. H. Schwartz, T. H. Aldrich, and M. E. Furth. "Abundant expression of ras proteins in Aplysia neurons." Journal of Cell Biology 103, no. 2 (August 1, 1986): 485–92. http://dx.doi.org/10.1083/jcb.103.2.485.
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We have cloned a DNA fragment from the marine mollusc Aplysia californica, which contains sequences homologous to mammalian ras genes, by screening a genomic library with a viral Ha-ras oncogene probe under conditions of low stringency hybridization. Nucleotide sequencing revealed a putative exon that encodes amino acids sharing 68% homology with residues 5 to 54 of mammalian p21ras polypeptides, and which therefore is likely to encode a ras-like Aplysia protein. The cloned locus, designated Apl-ras, is distinct from the Aplysia rho (ras-homologue) gene and appears to be more closely related to mammalian ras. We used a panel of monoclonal antibodies raised against v-Ha-ras p21 to precipitate an Mr 21,000 protein from extracts of Aplysia nervous tissue, ovotestis, and, to a much lesser degree, buccal muscle. Fluorescence immunocytochemistry revealed that ras-like protein is most abundant in neuronal cell bodies and axon processes, with staining most prominent at plasma membranes. Much less was present in other tissues. The prominence of ras protein in neurons, which are terminally differentiated and non-proliferating, indicates that the control of cell division is not the sole function of this proto-oncogene. The large identified neurons of Aplysia offer the opportunity to examine how ras protein might function in mature nerve cells.
3
Lowe, D. G., and D. V. Goeddel. "Heterologous expression and characterization of the human R-ras gene product." Molecular and Cellular Biology 7, no. 8 (August 1987): 2845–56. http://dx.doi.org/10.1128/mcb.7.8.2845-2856.1987.
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We directly expressed human R-ras 23,000-dalton protein (p23) cDNA in Escherichia coli under the control of the trp promoter. GTP-dependent phosphorylation of a p23 threonine 85 substitution mutant was observed. This result is in direct analogy to the autokinase activity of H-ras and K-ras threonine 59 substitution mutants. Normal p23 protein was detected in the human fibrosarcoma cell line HT1080 by immunoprecipitation with rabbit antibodies raised against an E. coli-expressed R-ras fusion protein. The R-ras p23 protein was found to be 3H labeled in the presence of [9,10(n)-3H]palmitic acid and is associated with the P100 membrane fraction of HT1080 cells. These data suggest that human R-ras p23 has biochemical properties very similar to those of the p21 products of the H-, K-, and N-ras proto-oncogenes. We constructed an R-ras minigene and engineered the expression of normal and mutant alleles from the simian virus 40 early region promoter. Normal and mutant R-ras gene products were authenticated by transient expression in COS-7 cells and immunoprecipitation. The valine 38-substituted R-ras p23 displayed reduced electrophoretic mobility. R-ras p21-like proteins, made by eliminating the first 26 R-ras codons, displayed evident mobility differences between the pro form and mature form, along with a valine 12 substitution-dependent change in electrophoretic mobility. Rat-1 fibroblasts were transfected with normal and mutant R-ras alleles and normal and activated H-ras alleles. Unlike the human T24 bladder oncogene-encoded p21, mutant R-ras alleles do not cause monolayer focus formation or growth in soft agar of rat fibroblasts.
4
Lowe, D. G., and D. V. Goeddel. "Heterologous expression and characterization of the human R-ras gene product." Molecular and Cellular Biology 7, no. 8 (August 1987): 2845–56. http://dx.doi.org/10.1128/mcb.7.8.2845.
Full textAbstract:
We directly expressed human R-ras 23,000-dalton protein (p23) cDNA in Escherichia coli under the control of the trp promoter. GTP-dependent phosphorylation of a p23 threonine 85 substitution mutant was observed. This result is in direct analogy to the autokinase activity of H-ras and K-ras threonine 59 substitution mutants. Normal p23 protein was detected in the human fibrosarcoma cell line HT1080 by immunoprecipitation with rabbit antibodies raised against an E. coli-expressed R-ras fusion protein. The R-ras p23 protein was found to be 3H labeled in the presence of [9,10(n)-3H]palmitic acid and is associated with the P100 membrane fraction of HT1080 cells. These data suggest that human R-ras p23 has biochemical properties very similar to those of the p21 products of the H-, K-, and N-ras proto-oncogenes. We constructed an R-ras minigene and engineered the expression of normal and mutant alleles from the simian virus 40 early region promoter. Normal and mutant R-ras gene products were authenticated by transient expression in COS-7 cells and immunoprecipitation. The valine 38-substituted R-ras p23 displayed reduced electrophoretic mobility. R-ras p21-like proteins, made by eliminating the first 26 R-ras codons, displayed evident mobility differences between the pro form and mature form, along with a valine 12 substitution-dependent change in electrophoretic mobility. Rat-1 fibroblasts were transfected with normal and mutant R-ras alleles and normal and activated H-ras alleles. Unlike the human T24 bladder oncogene-encoded p21, mutant R-ras alleles do not cause monolayer focus formation or growth in soft agar of rat fibroblasts.
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Mandanas, RA, DS Leibowitz, K. Gharehbaghi, T. Tauchi, GS Burgess, K. Miyazawa, HN Jayaram, and HS Boswell. "Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells." Blood 82, no. 6 (September 15, 1993): 1838–47. http://dx.doi.org/10.1182/blood.v82.6.1838.1838.
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Abstract The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth- factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13–259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G- protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.
6
Mandanas, RA, DS Leibowitz, K. Gharehbaghi, T. Tauchi, GS Burgess, K. Miyazawa, HN Jayaram, and HS Boswell. "Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells." Blood 82, no. 6 (September 15, 1993): 1838–47. http://dx.doi.org/10.1182/blood.v82.6.1838.bloodjournal8261838.
Full textAbstract:
The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth- factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13–259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G- protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.
7
Peace, D. J., W. Chen, H. Nelson, and M. A. Cheever. "T cell recognition of transforming proteins encoded by mutated ras proto-oncogenes." Journal of Immunology 146, no. 6 (March 15, 1991): 2059–65. http://dx.doi.org/10.4049/jimmunol.146.6.2059.
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Abstract Activated ras proto-oncogenes contribute to the pathogenesis of many animal and human malignancies. ras proto-oncogenes are generally activated by point mutations within codons 12 or 61, which result in the expression of ras protein (p21) bearing characteristic single amino acid substitutions at the corresponding residues. The purpose of the current study was to determine whether the presence of single transforming amino acid substitutions can render normal ras protein immunogenic and, thus, a possible target for T cell-mediated tumor therapy. In initial experiments, C57BL/6 mice were immunized with a synthetic peptide corresponding to residues 5 through 16 of p21 containing the transforming substitution of arginine for normal glycine at residue 12. The results demonstrated that class II MHC-restricted T cells which were specific for the peptide could be elicited, and that the peptide-induced T cells could specifically recognize the corresponding intact p21 ras protein. Recognition of p21 ras protein by peptide-specific T cells implies that C57BL/6 APC can process the activated ras protein in a fashion that allows presentation of digested protein by class II MHC molecules in a configuration similar to the configuration with synthetic peptide. Evaluation of the immunogenicity of peptides containing alternative transforming amino acid substitutions of ras protein demonstrated that some, but not all, were immunogenic in individual strains of mice. Therefore, although ras protein-specific T cells can be elicited by immunization with synthetic peptides, not all of the potential ras mutations commonly associated with malignancy may be recognizable by T cells from all individuals.
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McCoy, M. S., and R. A. Weinberg. "A human Ki-ras oncogene encodes two transforming p21 proteins." Molecular and Cellular Biology 6, no. 4 (April 1986): 1326–28. http://dx.doi.org/10.1128/mcb.6.4.1326-1328.1986.
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The human Ki-ras gene was previously reported to contain two alternative fourth exons which encode two distinct p21 proteins differing only at their carboxy termini. The present study shows that either p21 protein is able on its own to transform NIH 3T3 cells to a tumorigenic state.
9
McCoy, M. S., and R. A. Weinberg. "A human Ki-ras oncogene encodes two transforming p21 proteins." Molecular and Cellular Biology 6, no. 4 (April 1986): 1326–28. http://dx.doi.org/10.1128/mcb.6.4.1326.
Full textAbstract:
The human Ki-ras gene was previously reported to contain two alternative fourth exons which encode two distinct p21 proteins differing only at their carboxy termini. The present study shows that either p21 protein is able on its own to transform NIH 3T3 cells to a tumorigenic state.
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Sadler, S. E., J. L. Maller, and J. B. Gibbs. "Transforming ras proteins accelerate hormone-induced maturation and stimulate cyclic AMP phosphodiesterase in Xenopus oocytes." Molecular and Cellular Biology 10, no. 4 (April 1990): 1689–96. http://dx.doi.org/10.1128/mcb.10.4.1689-1696.1990.
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Transforming Harvey (Ha) ras oncogene products accelerated the time course of Xenopus oocyte maturation induced by insulin, insulinlike growth factor 1, or progesterone. The transforming constructs, [Val-12]Ha p21 and [Val-12, Thr-59]Ha p21, displayed equal potency and efficacy in their abilities to accelerate the growth peptide-induced response. Normal Ha p21 was only 60% as powerful and one-fifth as potent as the mutants containing valine in the 12 position. In contrast, two nontransforming constructs, [Val-12, Ala-35, Leu-36, Thr-59]Ha p21 and [Val-12, Thr-59]Ha(term-174) p21, had no effect on the time course of hormone-induced maturation. Effects of the transforming ras proteins on hormone-induced maturation correlated with their abilities to stimulate in vivo phosphodiesterase activity measured after microinjection of 200 microM cyclic [3H] AMP. When p21 injection followed 90 min of insulin treatment, there was no increase in phosphodiesterase activity over that measured after hormone treatment or p21 injection alone, but additive effects of p21 and insulin on enzyme activity were observed during the first 90 min of insulin treatment. Even though normal Ha p21 and transforming [Val-12, Thr-59]Ha p21 stimulated oocyte phosphodiesterase to equal levels when coinjected with substrate at the initiation of the in vivo assay, the transforming protein elicited a more sustained stimulation of enzyme activity. These results suggest that stimulation of a cyclic AMP phosphodiesterase activity associated with insulin-induced maturation is involved in the growth-promoting actions of ras oncogene products in Xenopus oocytes.
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Sadler, S. E., J. L. Maller, and J. B. Gibbs. "Transforming ras proteins accelerate hormone-induced maturation and stimulate cyclic AMP phosphodiesterase in Xenopus oocytes." Molecular and Cellular Biology 10, no. 4 (April 1990): 1689–96. http://dx.doi.org/10.1128/mcb.10.4.1689.
Full textAbstract:
Transforming Harvey (Ha) ras oncogene products accelerated the time course of Xenopus oocyte maturation induced by insulin, insulinlike growth factor 1, or progesterone. The transforming constructs, [Val-12]Ha p21 and [Val-12, Thr-59]Ha p21, displayed equal potency and efficacy in their abilities to accelerate the growth peptide-induced response. Normal Ha p21 was only 60% as powerful and one-fifth as potent as the mutants containing valine in the 12 position. In contrast, two nontransforming constructs, [Val-12, Ala-35, Leu-36, Thr-59]Ha p21 and [Val-12, Thr-59]Ha(term-174) p21, had no effect on the time course of hormone-induced maturation. Effects of the transforming ras proteins on hormone-induced maturation correlated with their abilities to stimulate in vivo phosphodiesterase activity measured after microinjection of 200 microM cyclic [3H] AMP. When p21 injection followed 90 min of insulin treatment, there was no increase in phosphodiesterase activity over that measured after hormone treatment or p21 injection alone, but additive effects of p21 and insulin on enzyme activity were observed during the first 90 min of insulin treatment. Even though normal Ha p21 and transforming [Val-12, Thr-59]Ha p21 stimulated oocyte phosphodiesterase to equal levels when coinjected with substrate at the initiation of the in vivo assay, the transforming protein elicited a more sustained stimulation of enzyme activity. These results suggest that stimulation of a cyclic AMP phosphodiesterase activity associated with insulin-induced maturation is involved in the growth-promoting actions of ras oncogene products in Xenopus oocytes.
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Lacal, J. C., and S. A. Aaronson. "Monoclonal antibody Y13-259 recognizes an epitope of the p21 ras molecule not directly involved in the GTP-binding activity of the protein." Molecular and Cellular Biology 6, no. 4 (April 1986): 1002–9. http://dx.doi.org/10.1128/mcb.6.4.1002-1009.1986.
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The p21 products of ras proto-oncogenes are GTP-binding proteins with associated GTPase activity. Recent studies have indicated that ras p21 may be required for the initiation of normal cell DNA synthesis, since microinjection of a monoclonal antibody, Y13-259, blocks serum stimulation of DNA synthesis in quiescent cell cultures (L. S. Mulcahy, M.R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We localized the structural domain within the p21 molecule recognized by the Y13-259 monoclonal antibody. By analysis of a series of bacterially expressed p21 deletion mutants, the monoclonal antibody was found to interact with a region between positions 70 and 89 in the p21 amino acid sequence. By comparison of the coding sequences of different p21 proteins recognized by this monoclonal antibody, a highly conserved amino acid region between positions 70 and 81 was found to be the most likely site for the epitope detected by the Y13-259 antibody. This monoclonal antibody was further shown not to interfere directly with in vitro biochemical functions of the molecule, including GTP binding, GTPase, and autokinase activities.
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Lacal, J. C., and S. A. Aaronson. "Monoclonal antibody Y13-259 recognizes an epitope of the p21 ras molecule not directly involved in the GTP-binding activity of the protein." Molecular and Cellular Biology 6, no. 4 (April 1986): 1002–9. http://dx.doi.org/10.1128/mcb.6.4.1002.
Full textAbstract:
The p21 products of ras proto-oncogenes are GTP-binding proteins with associated GTPase activity. Recent studies have indicated that ras p21 may be required for the initiation of normal cell DNA synthesis, since microinjection of a monoclonal antibody, Y13-259, blocks serum stimulation of DNA synthesis in quiescent cell cultures (L. S. Mulcahy, M.R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We localized the structural domain within the p21 molecule recognized by the Y13-259 monoclonal antibody. By analysis of a series of bacterially expressed p21 deletion mutants, the monoclonal antibody was found to interact with a region between positions 70 and 89 in the p21 amino acid sequence. By comparison of the coding sequences of different p21 proteins recognized by this monoclonal antibody, a highly conserved amino acid region between positions 70 and 81 was found to be the most likely site for the epitope detected by the Y13-259 antibody. This monoclonal antibody was further shown not to interfere directly with in vitro biochemical functions of the molecule, including GTP binding, GTPase, and autokinase activities.
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Clanton, D. J., Y. Y. Lu, D. G. Blair, and T. Y. Shih. "Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis." Molecular and Cellular Biology 7, no. 9 (September 1987): 3092–97. http://dx.doi.org/10.1128/mcb.7.9.3092-3097.1987.
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Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.
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Clanton, D. J., Y. Y. Lu, D. G. Blair, and T. Y. Shih. "Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis." Molecular and Cellular Biology 7, no. 9 (September 1987): 3092–97. http://dx.doi.org/10.1128/mcb.7.9.3092.
Full textAbstract:
Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.
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Litz-Jackson, S., AH Miller, GS Burgess, and HS Boswell. "Dissociation of nuclear events on p21 RAS transformation of FDC-P1 myeloid cells: c-jun/AP-1 expression versus c-myc transcription." Blood 79, no. 9 (May 1, 1992): 2404–14. http://dx.doi.org/10.1182/blood.v79.9.2404.2404.
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Abstract We have previously reported transformation to growth factor-independent proliferation in the interleukin-3 (IL-3)-dependent cell line FDC-P1 by high-level expression of the valine 12 Harvey RAS oncogene, following from a nonautocrine mechanism. The present study was undertaken to examine nuclear tertiary messenger, transcriptional response gene expression to deduce the intracellular signaling pathways responsible for this autonomous proliferation. We confirmed other reports that transformed p21RAS-expressing cells constitutively express the transcription factor complex jun/AP-1, in this case resulting from the ongoing expression of the c-jun and c-fos genes in the absence of IL-3. However, the ongoing growth factor independent expression of c-myc by a transcriptional mechanism in FDC-P1 cells expressing p21 RAS cannot be explained by intracellular signaling in the jun/AP-1 (protein kinase C) pathway. This conclusion derives from the observation that c-jun expression mediated via protein kinase C activation with phorbol ester (12–0-tetra decanoylphorbol-13-acetate, TPA) treatment does not lead to c-myc expression in parent FDC-P1 cells. On the contrary, FDC-P1 cells stably transfected with a c-myc gene controlled under the influence of a metallothionein IIA promoter (containing the TPA-responsive element [TRE]) express the transfected MTIIA-c-myc and downregulate the endogenous c-myc in response to protein kinase C activation with TPA. Further, nuclear proteins derived from cells expressing p21 RAS, which bind specifically to the purified c-myc P2 promoter, are not competed in their binding to the motif-rich P2 element by AP-1 oligonucleotide. Therefore, expression of the Harvey RAS oncogene in FDC-P1 myeloid cells leads to at least two pathways of cytoplasmic signaling. One pathway involves protein kinase C and c-jun/AP-1, but another pathway that is protein kinase C-independent appears to mediate c-myc transcription.
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Litz-Jackson, S., AH Miller, GS Burgess, and HS Boswell. "Dissociation of nuclear events on p21 RAS transformation of FDC-P1 myeloid cells: c-jun/AP-1 expression versus c-myc transcription." Blood 79, no. 9 (May 1, 1992): 2404–14. http://dx.doi.org/10.1182/blood.v79.9.2404.bloodjournal7992404.
Full textAbstract:
We have previously reported transformation to growth factor-independent proliferation in the interleukin-3 (IL-3)-dependent cell line FDC-P1 by high-level expression of the valine 12 Harvey RAS oncogene, following from a nonautocrine mechanism. The present study was undertaken to examine nuclear tertiary messenger, transcriptional response gene expression to deduce the intracellular signaling pathways responsible for this autonomous proliferation. We confirmed other reports that transformed p21RAS-expressing cells constitutively express the transcription factor complex jun/AP-1, in this case resulting from the ongoing expression of the c-jun and c-fos genes in the absence of IL-3. However, the ongoing growth factor independent expression of c-myc by a transcriptional mechanism in FDC-P1 cells expressing p21 RAS cannot be explained by intracellular signaling in the jun/AP-1 (protein kinase C) pathway. This conclusion derives from the observation that c-jun expression mediated via protein kinase C activation with phorbol ester (12–0-tetra decanoylphorbol-13-acetate, TPA) treatment does not lead to c-myc expression in parent FDC-P1 cells. On the contrary, FDC-P1 cells stably transfected with a c-myc gene controlled under the influence of a metallothionein IIA promoter (containing the TPA-responsive element [TRE]) express the transfected MTIIA-c-myc and downregulate the endogenous c-myc in response to protein kinase C activation with TPA. Further, nuclear proteins derived from cells expressing p21 RAS, which bind specifically to the purified c-myc P2 promoter, are not competed in their binding to the motif-rich P2 element by AP-1 oligonucleotide. Therefore, expression of the Harvey RAS oncogene in FDC-P1 myeloid cells leads to at least two pathways of cytoplasmic signaling. One pathway involves protein kinase C and c-jun/AP-1, but another pathway that is protein kinase C-independent appears to mediate c-myc transcription.
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Kalpokas, Irene, María Noel Martínez, Daniel Cavestany, Fernando Perdigón, Rodrigo Costa Mattos, and Ana Meikle. "Equine early pregnancy endocrine profiles and ipsilateral endometrial immune cell, gene expression and protein localisation response." Reproduction, Fertility and Development 33, no. 6 (2021): 410. http://dx.doi.org/10.1071/rd21001.
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We investigated the early effects of the equine embryo on maternal serum concentrations of insulin-like growth factor 1 (IGF1), leptin and adiponectin, uterine immune cells and genes and proteins related to embryo development and the maintenance of pregnancy. Ipsilateral endometrial expression was assessed on Days 7 and 13 after ovulation for the following transcripts: oestrogen receptor ERα (ESR1), progesterone receptor (PGR), progestin and adipoQ receptor family member 5 (PAQR5), oxytocin receptor (OXTR), prostaglandin-endoperoxide synthase 2 (PTGS2), raf-1 proto-oncogene serine/threonine kinase (RAF1), p21-activated kinase 6 (PAK6), fibroblast growth factor family member 9 (FGF9), IGF1 and its receptor (IGF1R), mucin 1 (MUC1), osteopontin (OPN), leptin receptor (LEPR) and adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2). Ipsilateral endometrial immunological cell infiltration and immunohistochemical protein localisation were evaluated on Days 7, 10 and 13 after ovulation for ERα, PGR, OXTR, PTGS2, IGF1, IGF1R, IGF2 and MUC1. Serum hormone concentrations were not affected by reproductive status. Pregnancy downregulated ESR1 and PGR mRNA levels, upregulated the expression of all other genes and affected the expression of all genes, except PGR, on Day 7 (compared with eight genes affected at Day 13). Proteins were affected by pregnancy or by its interaction with other variables (day of extraction and endometrial compartment). Pregnant mares had a higher lymphocyte count, which decreased towards Day 13. The effect of pregnancy on leucocytes and proteins was more evident in superficial endometrial compartments. The results of this study suggest that the equine embryo exerts prompt paracrine regulation of critical biological processes.
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Avraham, H., and R. A. Weinberg. "Characterization and expression of the human rhoH12 gene product." Molecular and Cellular Biology 9, no. 5 (May 1989): 2058–66. http://dx.doi.org/10.1128/mcb.9.5.2058-2066.1989.
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The rho genes constitute an evolutionarily conserved family having significant homology to the ras oncogene family. These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rat, and human; their 21,000-dalton products show strong conservation of structure. In humans, three classes of rho cDNA clones have been identified which differ by virtue of the presence of variable C-terminal domains: rhoH12, rhoH6, and rhoH9. The predicted 193 amino acids of human rhoH12 protein show 88% similarity with those of the human rhoH6 clone, 96.8% similarity with those of the Aplysia rho product, and 81.8% similarity with those of the yeast RHO1 protein. Rat-1 and NIH 3T3 mouse fibroblasts were transfected with clones containing the normal human rhoH12 allele as well as the variants encoding valine in place of the glycine and leucine in place of the glutamine normally found at residues 14 and 64, respectively. These replacements mirror the changes responsible for oncogenic activation of the related ras-encoded p21 proteins. These mutant rhoH12 clone alleles did not cause focus formation in monolayers or growth in soft agar. However, amplification of normal rhoH12 via cotransfection with a dihydrofolate reductase gene resulted in colonies that displayed reduced dependence on serum for growth, grew to higher saturation densities, and were tumorigenic when inoculated into nude mice. Normal p21rho protein was detected in the transfected cell lines as well as in normal cell lines by Western immunoblot and immunoprecipitation analysis with rabbit antibodies raised against the peptide corresponding to amino acids 122 to 135.
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Avraham, H., and R. A. Weinberg. "Characterization and expression of the human rhoH12 gene product." Molecular and Cellular Biology 9, no. 5 (May 1989): 2058–66. http://dx.doi.org/10.1128/mcb.9.5.2058.
Full textAbstract:
The rho genes constitute an evolutionarily conserved family having significant homology to the ras oncogene family. These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rat, and human; their 21,000-dalton products show strong conservation of structure. In humans, three classes of rho cDNA clones have been identified which differ by virtue of the presence of variable C-terminal domains: rhoH12, rhoH6, and rhoH9. The predicted 193 amino acids of human rhoH12 protein show 88% similarity with those of the human rhoH6 clone, 96.8% similarity with those of the Aplysia rho product, and 81.8% similarity with those of the yeast RHO1 protein. Rat-1 and NIH 3T3 mouse fibroblasts were transfected with clones containing the normal human rhoH12 allele as well as the variants encoding valine in place of the glycine and leucine in place of the glutamine normally found at residues 14 and 64, respectively. These replacements mirror the changes responsible for oncogenic activation of the related ras-encoded p21 proteins. These mutant rhoH12 clone alleles did not cause focus formation in monolayers or growth in soft agar. However, amplification of normal rhoH12 via cotransfection with a dihydrofolate reductase gene resulted in colonies that displayed reduced dependence on serum for growth, grew to higher saturation densities, and were tumorigenic when inoculated into nude mice. Normal p21rho protein was detected in the transfected cell lines as well as in normal cell lines by Western immunoblot and immunoprecipitation analysis with rabbit antibodies raised against the peptide corresponding to amino acids 122 to 135.
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Hattori, S., D. J. Clanton, T. Satoh, S. Nakamura, Y. Kaziro, M. Kawakita, and T. Y. Shih. "Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange." Molecular and Cellular Biology 7, no. 5 (May 1987): 1999–2002. http://dx.doi.org/10.1128/mcb.7.5.1999-2002.1987.
Full textAbstract:
The neutralizing monoclonal antibody Y13-259 severely hampers the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides but does not interfere with the association of GDP to p21. These results suggest that the nucleotide exchange reaction is critical for p21 function. Interestingly, the v-ras p21 has a much faster dissociation rate than the p21 of the c-ras proto-oncogene.
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Hattori, S., D. J. Clanton, T. Satoh, S. Nakamura, Y. Kaziro, M. Kawakita, and T. Y. Shih. "Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange." Molecular and Cellular Biology 7, no. 5 (May 1987): 1999–2002. http://dx.doi.org/10.1128/mcb.7.5.1999.
Full textAbstract:
The neutralizing monoclonal antibody Y13-259 severely hampers the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides but does not interfere with the association of GDP to p21. These results suggest that the nucleotide exchange reaction is critical for p21 function. Interestingly, the v-ras p21 has a much faster dissociation rate than the p21 of the c-ras proto-oncogene.
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Pan, B. T., and G. M. Cooper. "Role of phosphatidylinositide metabolism in ras-induced Xenopus oocyte maturation." Molecular and Cellular Biology 10, no. 3 (March 1990): 923–29. http://dx.doi.org/10.1128/mcb.10.3.923-929.1990.
Full textAbstract:
Microinjection of Xenopus oocytes with ras protein (p21) was used to investigate the role of phospholipid metabolism in ras-induced meiotic maturation. Induction of meiosis by ras was compared with induction by progesterone, insulin, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Neomycin, which specifically binds to phosphatidylinositides and inhibits their metabolism, blocked meiotic maturation induced by ras or insulin but not by progesterone or TPA. In addition, p21 and TPA, but not insulin or progesterone, stimulated the incorporation of 32Pi into oocyte lipids. ras protein specifically stimulated 32P incorporation into phosphatidylinositides, whereas both ras and TPA stimulated 32P incorporation into phosphatidylcholine and phosphatidylethanolamine. The stimulatory effect of p21 on phosphatidylinositide metabolism correlated with the dose response and kinetics of ras-induced meiotic maturation. In addition, the ras oncogene protein was more potent than the proto-oncogene protein both in inducing meiotic maturation and in stimulating phosphatidylinositide metabolism. These results indicate that phosphatidylinositide turnover is required for ras-induced meiosis and suggest that phosphatidylinositide-derived second messengers mediate the biological activity of ras in Xenopus oocytes.
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Pan, B. T., and G. M. Cooper. "Role of phosphatidylinositide metabolism in ras-induced Xenopus oocyte maturation." Molecular and Cellular Biology 10, no. 3 (March 1990): 923–29. http://dx.doi.org/10.1128/mcb.10.3.923.
Full textAbstract:
Microinjection of Xenopus oocytes with ras protein (p21) was used to investigate the role of phospholipid metabolism in ras-induced meiotic maturation. Induction of meiosis by ras was compared with induction by progesterone, insulin, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Neomycin, which specifically binds to phosphatidylinositides and inhibits their metabolism, blocked meiotic maturation induced by ras or insulin but not by progesterone or TPA. In addition, p21 and TPA, but not insulin or progesterone, stimulated the incorporation of 32Pi into oocyte lipids. ras protein specifically stimulated 32P incorporation into phosphatidylinositides, whereas both ras and TPA stimulated 32P incorporation into phosphatidylcholine and phosphatidylethanolamine. The stimulatory effect of p21 on phosphatidylinositide metabolism correlated with the dose response and kinetics of ras-induced meiotic maturation. In addition, the ras oncogene protein was more potent than the proto-oncogene protein both in inducing meiotic maturation and in stimulating phosphatidylinositide metabolism. These results indicate that phosphatidylinositide turnover is required for ras-induced meiosis and suggest that phosphatidylinositide-derived second messengers mediate the biological activity of ras in Xenopus oocytes.
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Zullo, J. N., and D. V. Faller. "P21 v-ras inhibits induction of c-myc and c-fos expression by platelet-derived growth factor." Molecular and Cellular Biology 8, no. 12 (December 1988): 5080–85. http://dx.doi.org/10.1128/mcb.8.12.5080-5085.1988.
Full textAbstract:
The viral oncogene v-ras inhibited the platelet-derived growth factor (PDGF)-induced upregulation of c-myc and c-fos proto-oncogene expression in fibroblast monolayers. These v-ras-containing cells proliferated in the absence of c-myc induction and no longer required PDGF to support growth. Fibroblasts expressing v-ras continued to express the same number of functional PDGF receptors on their surface as uninfected cells, yet the usual induction of transcription of the genes c-myc, c-fos, and JE in response to PDGF stimulation did not occur in the presence of newly introduced v-ras or chronic v-ras gene expression, and synthesis of c-myc protein did not occur. This inhibitory effect on growth factor-mediated induction of cellular proto-oncogenes was specific for PDGF in that induction of the c-myc and c-fos genes by certain other factors was not impaired.
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Zullo, J. N., and D. V. Faller. "P21 v-ras inhibits induction of c-myc and c-fos expression by platelet-derived growth factor." Molecular and Cellular Biology 8, no. 12 (December 1988): 5080–85. http://dx.doi.org/10.1128/mcb.8.12.5080.
Full textAbstract:
The viral oncogene v-ras inhibited the platelet-derived growth factor (PDGF)-induced upregulation of c-myc and c-fos proto-oncogene expression in fibroblast monolayers. These v-ras-containing cells proliferated in the absence of c-myc induction and no longer required PDGF to support growth. Fibroblasts expressing v-ras continued to express the same number of functional PDGF receptors on their surface as uninfected cells, yet the usual induction of transcription of the genes c-myc, c-fos, and JE in response to PDGF stimulation did not occur in the presence of newly introduced v-ras or chronic v-ras gene expression, and synthesis of c-myc protein did not occur. This inhibitory effect on growth factor-mediated induction of cellular proto-oncogenes was specific for PDGF in that induction of the c-myc and c-fos genes by certain other factors was not impaired.
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Senapedis, William, Ryan George, Dilara McCauley, Joel Ellis, Marsha Crochiere, Michael Savona, Sharon Shacham, Yosef Landesman, and Erkan Baloglu. "Novel Selective Orally Bioavailable Small Molecule PAK4 Allosteric Modulators (PAMs) Display Anti-Tumor Activity in Vitro and in Vivo in Hematological Malignancies." Blood 124, no. 21 (December 6, 2014): 2208. http://dx.doi.org/10.1182/blood.v124.21.2208.2208.
Full textAbstract:
Abstract Introduction: Many hematological cancers have been successfully treated through identification of specialized targets in each specific tumor subtype (e.g. BTK inhibition in NHL or proteasome inhibition in multiple myeloma). The p21-Activated Kinase 4 (PAK4) is critical to cellular signaling and may represent a new target for therapy in many hematologic malignancies. PAK4 is a member of the PAK family of proteins that regulate cell survival, cell division and apoptosis. The six members of the PAK family are divided into two groups; Group I (PAK1, 2, 3) and Group II (PAK4, 5, 6), based upon their sequence homology and regulatory mechanisms. PAK4 is a member of the group II family of PAKs and is amplified or mutated in many cancer types. PAK4 is also a key downstream effector of the K-Ras pathway. Methods: Flow cytometry and CellTiter AQueous One (MTS) assays were used to determine compound effects on cell cycle distribution, proliferation and viability. Immunoblots were used to measure effects of compounds on protein steady state levels and phosphorylation. The T-cell ALL cell line, MOLT-4, and the mantle cell lymphoma cell line, Z-138, were used in xenograft models in mice to test the in vivo efficacy of these compounds. Results: We have identified selective, orally bioavailable, small molecule PAK4 allosteric modulators {PAMs; e.g. KPT-8752 (mw: 585.6), KPT-9274 (mw: 610.6), and KPT-9331 (mw: 628.6)} which demonstrated selective anti-tumor activity in a variety of hematological cancer cell lines (IC50 values = 0.005 – 1 mM). Treatment of cancer cells with these small molecules resulted in the reduction of PAK4 steady state levels and reduced phosphorylation of key growth signaling proteins such as Akt, β-catenin, cofilin, p21, and cyclin D1. There was a measurable increase in phospho-AMPK indicative of autophagy and stress. These allosteric modulators induced apoptosis through the activation of caspases 3 and 8 and subsequent cleavage of PARP. In MOLT-4 and Z-138 xenograft mouse models, daily treatment with oral PAMs resulted in near elimination of small (100 mm3) and large (800 mm3) tumors in the absence of any clinical signs of toxicity within the animals. Additional cell line and primary tumor models are currently being explored. Conclusions: PAK4 represents a novel anti-cancer target as a major downstream effector of the Ras oncogene. We have identified selective, orally-bioavailable small molecule PAK4 allosteric modulators which induce potent cytotoxicity in multiple leukemia and lymphoma cell lines with minimal toxicity to normal cell in vitro and clear anti-tumor activity with excellent tolerability in in vivo models of hematological cancers. These compounds inactivate PAK4 by directly inducing PAK4 destabilization. This represents a novel mechanism of the protein kinase inactivation involving degradation of PAK4 rather than direct inhibition of the kinase activity. Based on the in vitro and in vivo activity, these PAK4 allosteric modulators show promising results for the treatment of a wide variety of hematological cancers. Disclosures Senapedis: Karyopharm: Employment. George:Karyopharm: Employment. McCauley:Karyopharm Therapeutics: Employment, Equity Ownership. Ellis:Karyopharm: Employment. Crochiere:Karyopharm: Employment. Savona:Karyopharm: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Shacham:Karyopharm Therapeutics: Employment. Landesman:Karyopharm Therapeutics: Employment. Baloglu:Karyopharm: Employment.
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Pulciani, S., E. Santos, L. K. Long, V. Sorrentino, and M. Barbacid. "ras gene Amplification and malignant transformation." Molecular and Cellular Biology 5, no. 10 (October 1985): 2836–41. http://dx.doi.org/10.1128/mcb.5.10.2836-2841.1985.
Full textAbstract:
Morphologic transformation of NIH 3T3 mouse cells occurs upon transfection of these cells with large amounts (greater than or equal to 10 micrograms) of recombinant DNA molecules carrying the normal human H-ras-1 proto-oncogene. We provide experimental evidence indicating that transformation of these NIH 3T3 cells results from the combined effect of multiple copies of the H-ras-1 proto-oncogene rather than from spontaneous mutation of one of the transfected H-ras-1 clones (E. Santos, E.P. Reddy, S. Pulciani, R.J. Feldman, and M. Barbacid, Proc. Natl. Acad. Sci. USA 80:4679-4683, 1983). Levels of H-ras-1 RNA and p21 expression are highly elevated in the NIH 3T3 transformants, and in those cases examined, these levels correlate with the malignant properties of these cells. We have also investigated the presence of amplified ras genes in a variety of human carcinomas. In 75 tumor biopsies, we found amplification of the human K-ras-2 locus in one carcinoma of the lung. These results indicate that ras gene amplification is an alternative pathway by which ras genes may participate in the development of human neoplasia.
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Pulciani, S., E. Santos, L. K. Long, V. Sorrentino, and M. Barbacid. "ras gene Amplification and malignant transformation." Molecular and Cellular Biology 5, no. 10 (October 1985): 2836–41. http://dx.doi.org/10.1128/mcb.5.10.2836.
Full textAbstract:
Morphologic transformation of NIH 3T3 mouse cells occurs upon transfection of these cells with large amounts (greater than or equal to 10 micrograms) of recombinant DNA molecules carrying the normal human H-ras-1 proto-oncogene. We provide experimental evidence indicating that transformation of these NIH 3T3 cells results from the combined effect of multiple copies of the H-ras-1 proto-oncogene rather than from spontaneous mutation of one of the transfected H-ras-1 clones (E. Santos, E.P. Reddy, S. Pulciani, R.J. Feldman, and M. Barbacid, Proc. Natl. Acad. Sci. USA 80:4679-4683, 1983). Levels of H-ras-1 RNA and p21 expression are highly elevated in the NIH 3T3 transformants, and in those cases examined, these levels correlate with the malignant properties of these cells. We have also investigated the presence of amplified ras genes in a variety of human carcinomas. In 75 tumor biopsies, we found amplification of the human K-ras-2 locus in one carcinoma of the lung. These results indicate that ras gene amplification is an alternative pathway by which ras genes may participate in the development of human neoplasia.
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Samid, D., D. M. Flessate, and R. M. Friedman. "Interferon-induced revertants of ras-transformed cells: resistance to transformation by specific oncogenes and retransformation by 5-azacytidine." Molecular and Cellular Biology 7, no. 6 (June 1987): 2196–200. http://dx.doi.org/10.1128/mcb.7.6.2196-2200.1987.
Full textAbstract:
Prolonged alpha/beta interferon (IFN-alpha/beta) treatment of NIH 3T3 cells transformed by a long terminal repeat-activated Ha-ras proto-oncogene resulted in revertants that maintained a nontransformed phenotype long after IFN treatment had been discontinued. Cloned persistent revertants (PRs) produced large amounts of the ras-encoded p21 and were refractile to transformation by EJras DNA and by transforming retroviruses which carried the v-Ha-ras, v-Ki-ras, v-abl, or v-fes oncogene. Transient treatment either in vitro or in vivo with cytidine analogs that alter gene expression by inhibiting DNA methylation resulted in transformation of PR, but not of NIH 3T3, cells. The PR retransformants reverted again with IFN, suggesting that DNA methylation is involved in IFN-induced persistent reversion.
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Samid, D., D. M. Flessate, and R. M. Friedman. "Interferon-induced revertants of ras-transformed cells: resistance to transformation by specific oncogenes and retransformation by 5-azacytidine." Molecular and Cellular Biology 7, no. 6 (June 1987): 2196–200. http://dx.doi.org/10.1128/mcb.7.6.2196.
Full textAbstract:
Prolonged alpha/beta interferon (IFN-alpha/beta) treatment of NIH 3T3 cells transformed by a long terminal repeat-activated Ha-ras proto-oncogene resulted in revertants that maintained a nontransformed phenotype long after IFN treatment had been discontinued. Cloned persistent revertants (PRs) produced large amounts of the ras-encoded p21 and were refractile to transformation by EJras DNA and by transforming retroviruses which carried the v-Ha-ras, v-Ki-ras, v-abl, or v-fes oncogene. Transient treatment either in vitro or in vivo with cytidine analogs that alter gene expression by inhibiting DNA methylation resulted in transformation of PR, but not of NIH 3T3, cells. The PR retransformants reverted again with IFN, suggesting that DNA methylation is involved in IFN-induced persistent reversion.
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Moura, Margarida M., Branca M. Cavaco, and Valeriano Leite. "RAS proto-oncogene in medullary thyroid carcinoma." Endocrine-Related Cancer 22, no. 5 (October 2015): R235—R252. http://dx.doi.org/10.1530/erc-15-0070.
Full textAbstract:
Medullary thyroid carcinoma (MTC) is a rare malignancy originating from the calcitonin-secreting parafollicular thyroid C cells. Approximately 75% of cases are sporadic. Rearranged during transfection (RET) proto-oncogene plays a crucial role in MTC development. BesidesRET, other oncogenes commonly involved in the pathogenesis of human cancers have also been investigated in MTC. The family of humanRASgenes includes the highly homologousHRAS,KRAS, andNRASgenes that encode three distinct proteins. Activating mutations in specific hotspots of theRASgenes are found in about 30% of all human cancers. In thyroid neoplasias,RASgene point mutations, mainly inNRAS, are detected in benign and malignant tumors arising from the follicular epithelium. However, recent reports have also describedRASmutations in MTC, namely inHRASandKRAS. Overall, the prevalence ofRASmutations in sporadic MTC varies between 0–43.3%, occurring usually in tumors with WTRETand rarely in those harboring aRETmutation, suggesting that activation of these proto-oncogenes represents alternative genetic events in sporadic MTC tumorigenesis. Thus, the assessment ofRASmutation status can be useful to define therapeutic strategies inRETWT MTC. MTC patients withRASmutations have an intermediate risk for aggressive cancer, between those withRETmutations in exons 15 and 16, which are associated with the worst prognosis, and cases with otherRETmutations, which have the most indolent course of the disease. Recent results from exome sequencing indicate that, besides mutations inRET,HRAS, andKRAS, no other recurrent driver mutations are present in MTC.
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Rapoport, M. J., L. Weiss, A. Mor, T. Bistritzer, Y. Ramot, and S. Slavin. "Prevention of autoimmune diabetes by linomide in nonobese diabetic (NOD) mice is associated with up-regulation of the TCR-mediated activation of p21(ras)." Journal of Immunology 157, no. 10 (November 15, 1996): 4721–25. http://dx.doi.org/10.4049/jimmunol.157.10.4721.
Full textAbstract:
Abstract Oral therapy with linomide protects prediabetic nonobese diabetic (NOD) mice from insulin-dependent diabetes mellitus. The mechanisms by which linomide exerts its protective effect are not fully understood. A decreased TCR-mediated activity of the GTP-GDP binding p21(ras) proto-oncogene is associated with prediabetes in NOD mice. However, the role of this signal transduction defect in the pathogenesis of autoimmune diabetes is not known. The TCR-mediated and protein kinase C-induced activations of p21(ras) were determined in mononuclear cells from lymph nodes of linomide-treated and untreated prediabetic NOD mice. TCR cross-linking by Con A induced an increase of 13 +/- 6.8% and a decrease of 0.8 +/- 1.8% in p21(ras) activity in the linomide-treated group and the untreated controls, respectively. Cell stimulation with PMA resulted in a 15 +/- 2% increase in p21(ras) activity in the linomide-treated mice and a 10 +/- 11.4% decrease in the untreated mice. Protein levels of p21(ras) and its regulatory elements, the GTPase-activating protein and the guanine nucleotide-releasing factor, mSOS, were comparable in both groups. We, therefore, conclude that prevention of autoimmune diabetes by linomide is associated with up-regulation of the p21(ras) T cell signal transduction defect in NOD mice.
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Tanikawa, Eiko, Osamu Mori, Hiroshi Hachisuka, and Yoichiro Sasai. "Expression of ras proto-oncogene related protein p21 in normal human skin and cutaneous tumours." Acta Histochemica 93, no. 1 (January 1992): 282–89. http://dx.doi.org/10.1016/s0065-1281(11)80225-4.
Full text
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Nandi, Sisir, and Manish C. Bagchi. "Exploring CDKs, Ras-ERK, and PI3K-Aktin Abnormal Signaling and Cancer." Journal of Cancer Research Updates 11 (October 27, 2022): 63–69. http://dx.doi.org/10.30683/1929-2279.2022.11.09.
Full textAbstract:
Cancer or malignancy can be defined as abnormal growth and cell division. Malignancies spread, through metastasis invasion, or implantation into distant sites by which cancer cells can move through the bloodstream or lymphatic system to distant locations. The body cells follow mitotic cell division process. Normal cell division occurs through the normal signal transduction through proto-oncogenes responsible for the cell proliferation and differentiation. Mutation of these proto-oncogene leads to oncogene which can modify the gene expression and function through abnormal signal transduction, making uncontrolled growth of cells. The mitotic cell cycle is regulated by the signal transduction through the cyclin dependent kinases (CDKs), Ras-ERK and PI3K-Akt.Abnormal signaling occurs through the mutation of these genes leading to the cancer. The present review shortly reported the role of these proteins in abnormal signal transduction and cancer.
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Miller, A. C., J. Gafner, E. P. Clark, and D. Samid. "Posttranscriptional down-regulation of ras oncogene expression by inhibitors of cellular glutathione." Molecular and Cellular Biology 13, no. 7 (July 1993): 4416–22. http://dx.doi.org/10.1128/mcb.13.7.4416-4422.1993.
Full textAbstract:
Alterations in intracellular glutathione (GSH) content are known to affect intrinsic responses to ionizing radiation. More recently, it became apparent that radiation responses may depend also on the expression of specific oncogenes, including ras. These findings, suggesting a possible link between GSH and ras, led us to examine the effect of various GSH modulators on ras expression. Treatment of c-Ha-ras-transformed NIH 3T3 cells with L-buthionine S'R'-sulfoximine, dimethylfumarate, or N',N'-1,3-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea resulted in dose- and time-dependent reduction in ras mRNA steady-state levels followed by a decrease in ras-encoded p21 protein production. The effect on ras correlated with the extent of GSH decline, was common to different members of the ras family, and was independent of the mode of oncogene activation or cell phenotype. Indeed, similar drug effects were observed with murine cells in which overexpression of the c-Ha-ras proto-oncogene was due to transcriptional activation (PR4, nontumorigenic) or gene amplification (NIH 136, tumorigenic) and with malignant cells expressing a mutated Ha-ras (RS504). Moreover, N-ras, EJras, and Ki-ras in human tumor cells were similarly affected. Molecular analysis revealed a significant decrease in ras mRNA half-life in cells subjected to GSH inhibition, an effect that required de novo protein synthesis, but there was no change in the rate of gene transcription. These results indicate that pharmacological manipulation of cellular GSH content can down-regulate ras expression at the posttranscriptional level by destabilizing ras transcripts. The potential clinical implications are discussed.
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Miller, A. C., J. Gafner, E. P. Clark, and D. Samid. "Posttranscriptional down-regulation of ras oncogene expression by inhibitors of cellular glutathione." Molecular and Cellular Biology 13, no. 7 (July 1993): 4416–22. http://dx.doi.org/10.1128/mcb.13.7.4416.
Full textAbstract:
Alterations in intracellular glutathione (GSH) content are known to affect intrinsic responses to ionizing radiation. More recently, it became apparent that radiation responses may depend also on the expression of specific oncogenes, including ras. These findings, suggesting a possible link between GSH and ras, led us to examine the effect of various GSH modulators on ras expression. Treatment of c-Ha-ras-transformed NIH 3T3 cells with L-buthionine S'R'-sulfoximine, dimethylfumarate, or N',N'-1,3-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea resulted in dose- and time-dependent reduction in ras mRNA steady-state levels followed by a decrease in ras-encoded p21 protein production. The effect on ras correlated with the extent of GSH decline, was common to different members of the ras family, and was independent of the mode of oncogene activation or cell phenotype. Indeed, similar drug effects were observed with murine cells in which overexpression of the c-Ha-ras proto-oncogene was due to transcriptional activation (PR4, nontumorigenic) or gene amplification (NIH 136, tumorigenic) and with malignant cells expressing a mutated Ha-ras (RS504). Moreover, N-ras, EJras, and Ki-ras in human tumor cells were similarly affected. Molecular analysis revealed a significant decrease in ras mRNA half-life in cells subjected to GSH inhibition, an effect that required de novo protein synthesis, but there was no change in the rate of gene transcription. These results indicate that pharmacological manipulation of cellular GSH content can down-regulate ras expression at the posttranscriptional level by destabilizing ras transcripts. The potential clinical implications are discussed.
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Durkin, J. P., and J. F. Whitfield. "The viral Ki-ras gene must be expressed in the G2 phase if ts Kirsten sarcoma virus-infected NRK cells are to proliferate in serum-free medium." Molecular and Cellular Biology 7, no. 1 (January 1987): 444–49. http://dx.doi.org/10.1128/mcb.7.1.444-449.1987.
Full textAbstract:
NRK cells infected with a temperature-sensitive Kirsten sarcoma virus (ts371 KSV) are transformed at 36 degrees C, but are untransformed at 41 degrees C which inactivates the abnormally thermolabile oncogenic p21Ki product of the viral Ki-ras gene. At 41 degrees C, tsKSV-infected NRK cells were arrested in G0/G1 when incubated in serum-free medium, but could then be stimulated to transit G1, replicate DNA, and divide by adding serum at 41 degrees C or dropping the temperature to a p21-activating 36 degrees C without adding serum. When quiescent cells at 41 degrees C were stimulated to transit G1 in serum-free medium by activating p21 at 36 degrees C and then shifted back to the p21-inactivating 41 degrees C in the mid-S phase, they continued replicating DNA but could not transit G2. Reactivating p21 in the G2-arrested cells by once again lowering the temperature to 36 degrees C stimulated a rapid entry into mitosis. By contrast, while serum-stimulated quiescent G0 cells at 41 degrees C replicate DNA and divide, serum did not induce G2-arrested cells to enter mitosis, indicating that serum growth factors may trigger events in the G1 phase that ultimately determine G2 transit. These observations made with the viral ras product suggest that cellular ras proto-oncogene products have a role in G2 transit of normal cells.
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Durkin, J. P., and J. F. Whitfield. "The viral Ki-ras gene must be expressed in the G2 phase if ts Kirsten sarcoma virus-infected NRK cells are to proliferate in serum-free medium." Molecular and Cellular Biology 7, no. 1 (January 1987): 444–49. http://dx.doi.org/10.1128/mcb.7.1.444.
Full textAbstract:
NRK cells infected with a temperature-sensitive Kirsten sarcoma virus (ts371 KSV) are transformed at 36 degrees C, but are untransformed at 41 degrees C which inactivates the abnormally thermolabile oncogenic p21Ki product of the viral Ki-ras gene. At 41 degrees C, tsKSV-infected NRK cells were arrested in G0/G1 when incubated in serum-free medium, but could then be stimulated to transit G1, replicate DNA, and divide by adding serum at 41 degrees C or dropping the temperature to a p21-activating 36 degrees C without adding serum. When quiescent cells at 41 degrees C were stimulated to transit G1 in serum-free medium by activating p21 at 36 degrees C and then shifted back to the p21-inactivating 41 degrees C in the mid-S phase, they continued replicating DNA but could not transit G2. Reactivating p21 in the G2-arrested cells by once again lowering the temperature to 36 degrees C stimulated a rapid entry into mitosis. By contrast, while serum-stimulated quiescent G0 cells at 41 degrees C replicate DNA and divide, serum did not induce G2-arrested cells to enter mitosis, indicating that serum growth factors may trigger events in the G1 phase that ultimately determine G2 transit. These observations made with the viral ras product suggest that cellular ras proto-oncogene products have a role in G2 transit of normal cells.
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Gulbins, E., K. M. Coggeshall, C. Langlet, G. Baier, N. Bonnefoy-Berard, P. Burn, A. Wittinghofer, S. Katzav, and A. Altman. "Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein." Molecular and Cellular Biology 14, no. 2 (February 1994): 906–13. http://dx.doi.org/10.1128/mcb.14.2.906-913.1994.
Full textAbstract:
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.
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Gulbins, E., K. M. Coggeshall, C. Langlet, G. Baier, N. Bonnefoy-Berard, P. Burn, A. Wittinghofer, S. Katzav, and A. Altman. "Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein." Molecular and Cellular Biology 14, no. 2 (February 1994): 906–13. http://dx.doi.org/10.1128/mcb.14.2.906.
Full textAbstract:
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.
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Kremer, N. E., G. D'Arcangelo, S. M. Thomas, M. DeMarco, J. S. Brugge, and S. Halegoua. "Signal transduction by nerve growth factor and fibroblast growth factor in PC12 cells requires a sequence of src and ras actions." Journal of Cell Biology 115, no. 3 (November 1, 1991): 809–19. http://dx.doi.org/10.1083/jcb.115.3.809.
Full textAbstract:
We have investigated the roles of pp60c-src and p21c-ras proteins in transducing the nerve growth factor (NGF) and fibroblast growth factor (FGF) signals which promote the sympathetic neuronlike phenotype in PC12 cells. Neutralizing antibodies directed against either Src or Ras proteins were microinjected into fused PC12 cells. Each antibody both prevented and reversed NGF- or FGF-induced neurite growth, a prominent morphological marker for the neuronal phenotype. These data demonstrate the involvement of both pp60c-src and p21c-ras proteins in NGF and FGF actions in PC12 cells, and establish a physiological role for the pp60c-src tyrosine kinase in signal transduction pathways initiated by receptor tyrosine kinases in these cells. Additional microinjection experiments, using PC12 transfectants containing inducible v-src or ras oncogene activities, demonstrated a specific sequence of Src and Ras actions. Microinjection of anti-Ras antibody blocked v-src-induced neurite growth, but microinjection of anti-Src antibodies had no effect on ras oncogene-induced neurite growth. We propose that a cascade of Src and Ras actions, with Src acting first, is a significant feature of the signal transduction pathways for NGF and FGF. The Src-Ras cascade may define a functional cassette in the signal transduction pathways used by growth factors and other ligands whose receptors have diverse structures and whose range of actions on various cell types include mitogenesis and differentiation.
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Nelson, Andrew C., Thomas J. Turbyville, Srisathiyanarayanan Dharmaiah, Megan Rigby, Rendong Yang, Ting-You Wang, John Columbus, et al. "RAS internal tandem duplication disrupts GTPase-activating protein (GAP) binding to activate oncogenic signaling." Journal of Biological Chemistry 295, no. 28 (May 11, 2020): 9335–48. http://dx.doi.org/10.1074/jbc.ra119.011080.
Full textAbstract:
The oncogene RAS is one of the most widely studied proteins in cancer biology, and mutant active RAS is a driver in many types of solid tumors and hematological malignancies. Yet the biological effects of different RAS mutations and the tissue-specific clinical implications are complex and nuanced. Here, we identified an internal tandem duplication (ITD) in the switch II domain of NRAS from a patient with extremely aggressive colorectal carcinoma. Results of whole-exome DNA sequencing of primary and metastatic tumors indicated that this mutation was present in all analyzed metastases and excluded the presence of any other clear oncogenic driver mutations. Biochemical analysis revealed increased interaction of the RAS ITD with Raf proto-oncogene Ser/Thr kinase (RAF), leading to increased phosphorylation of downstream MAPK/ERK kinase (MEK)/extracellular signal–regulated kinase (ERK). The ITD prevented interaction with neurofibromin 1 (NF1)–GTPase–activating protein (GAP), providing a mechanism for sustained activity of the RAS ITD protein. We present the first crystal structures of NRAS and KRAS ITD at 1.65–1.75 Å resolution, respectively, providing insight into the physical interactions of this class of RAS variants with its regulatory and effector proteins. Our in-depth bedside-to-bench analysis uncovers the molecular mechanism underlying a case of highly aggressive colorectal cancer and illustrates the importance of robust biochemical and biophysical approaches in the implementation of individualized medicine.
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Sassone-Corsi, P., C. J. Der, and I. M. Verma. "ras-induced neuronal differentiation of PC12 cells: possible involvement of fos and jun." Molecular and Cellular Biology 9, no. 8 (August 1989): 3174–83. http://dx.doi.org/10.1128/mcb.9.8.3174-3183.1989.
Full textAbstract:
Rat pheochromocytoma PC12 cells differentiate to sympathetic neuron-like cells upon treatment with nerve growth factor (NGF). The ras and src transforming proteins also induce PC12 neuronal differentiation and are likely to involve the protein kinase C signal transduction pathway. Using a number of ras mutants, we have established that the domains of oncogenic ras protein responsible for PC12 differentiation overlap those required for cellular transformation. All of the ras mutants that induced neuronal differentiation also activated c-fos transcription through the dyad symmetry element (DSE). Transforming ras protein activated an intracellular signal pathway, which led to the induction of 12-O-tetradecanoyl phorbol-13-acetate-responsive elements; activation was enhanced by coexpression of the proto-oncogene jun (encoding AP-1) and was further augmented by fos. Nuclear extracts from ras-infected PC12 cells showed an increased AP-1 DNA-binding activity. Transcriptional activation by ras was independent of the cyclic AMP-dependent pathway of signal transduction. We propose a possible involvement of fos and jun in ras-induced differentiation.
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Sassone-Corsi, P., C. J. Der, and I. M. Verma. "ras-induced neuronal differentiation of PC12 cells: possible involvement of fos and jun." Molecular and Cellular Biology 9, no. 8 (August 1989): 3174–83. http://dx.doi.org/10.1128/mcb.9.8.3174.
Full textAbstract:
Rat pheochromocytoma PC12 cells differentiate to sympathetic neuron-like cells upon treatment with nerve growth factor (NGF). The ras and src transforming proteins also induce PC12 neuronal differentiation and are likely to involve the protein kinase C signal transduction pathway. Using a number of ras mutants, we have established that the domains of oncogenic ras protein responsible for PC12 differentiation overlap those required for cellular transformation. All of the ras mutants that induced neuronal differentiation also activated c-fos transcription through the dyad symmetry element (DSE). Transforming ras protein activated an intracellular signal pathway, which led to the induction of 12-O-tetradecanoyl phorbol-13-acetate-responsive elements; activation was enhanced by coexpression of the proto-oncogene jun (encoding AP-1) and was further augmented by fos. Nuclear extracts from ras-infected PC12 cells showed an increased AP-1 DNA-binding activity. Transcriptional activation by ras was independent of the cyclic AMP-dependent pathway of signal transduction. We propose a possible involvement of fos and jun in ras-induced differentiation.
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Green, N. K., M. D. Gammage, J. A. Franklyn, and M. C. Sheppard. "Regulation by thyroid status of c-myc, c-fos and H-ras mRNAs in the rat myocardium." Journal of Endocrinology 130, no. 2 (August 1991): 239–44. http://dx.doi.org/10.1677/joe.0.1300239.
Full textAbstract:
ABSTRACT Effects of thyroid status on expression of a variety of myocardial genes, such as those encoding contractile proteins, have been reported, as well as interactions between thyroid hormones and developmental and haemodynamic regulation of contractile protein synthesis. In addition, it is clear that developmental and haemodynamic factors regulate expression of specific proto-oncogenes, including c-myc, c-fos and H-ras, in the myocardium but the effect of thyroid status on such proto-oncogene products, which are proposed to play a critical signal-transducing role in the heart, has been previously unexplored. In order to determine whether changes in thyroid status are associated with changes in expression of these putative intracellular signals, we examined the effect of hypothyroidism and tri-iodothyronine (T3) treatment on myocardial levels of c-myc, c-fos and H-ras mRNAs in the rat. The induction of hypothyroidism was associated with a marked increase in myocardial c-myc, c-fos and H-ras mRNAs, changes reversed by 72 h of T3 replacement. Administration of T3 to euthyroid rats had no significant effect on myocardial c-myc or c-fos mRNAs, but inhibition of H-ras mRNA by T3 was evident. These observations demonstrating influences of thyroid status on expression of specific proto-oncogenes suggest that thyroid hormones, as well as exerting direct effects on expression of functionally important myocardial genes, also interact with the cellular transduction pathways mediated by the products of the c-myc, c-fos and H-ras genes. Journal of Endocrinology (1991) 130, 239–244
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Gulbins, E., C. Langlet, G. Baier, N. Bonnefoy-Berard, E. Herbert, A. Altman, and K. M. Coggeshall. "Tyrosine phosphorylation and activation of Vav GTP/GDP exchange activity in antigen receptor-triggered B cells." Journal of Immunology 152, no. 5 (March 1, 1994): 2123–29. http://dx.doi.org/10.4049/jimmunol.152.5.2123.
Full textAbstract:
Abstract Ag receptor triggering in B cells stimulates the activity of receptor-associated tyrosine protein kinases (TPK), leading to tyrosine phosphorylation of several cellular substrates, one of which is the Vav proto-oncogene product. We have recently determined that Vav is a TPK-regulated guanine nucleotide exchange factor for Ras in T cells. Here, we show that B cell extracts or Vav immunoprecipitates contain a Ras GDP/GTP exchange activity that is stimulated upon surface Ig (slg) triggering. The receptor-mediated stimulation of Vav exchange activity was blocked by the TPK antagonist, herbimycin A. Furthermore, immunodepletion of Vav from the B cell extracts removed approximately 80% of the Ras GDP/GTP exchange activity. These findings indicate, first, that B cell-derived Vav possesses GDP/GTP exchange activity for Ras; second, that the exchange activity of Vav is accelerated by a slg-triggered, herbimycin A-sensitive TPK and, third, that Vav accounts for most of the receptor-stimulated Ras GDP/GTP exchange activity. Thus, Vav may serve as a critical component in slg-mediated signal transduction pathways by coupling receptor-associated TPK to the activation of Ras proteins.
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Shimizu, Takanobu, Takeshi Nakamura, Hironori Inaba, Hiroaki Iwasa, Junichi Maruyama, Kyoko Arimoto-Matsuzaki, Takao Nakata, Hiroshi Nishina, and Yutaka Hata. "The RAS-interacting chaperone UNC119 drives the RASSF6–MDM2–p53 axis and antagonizes RAS-mediated malignant transformation." Journal of Biological Chemistry 295, no. 32 (June 18, 2020): 11214–30. http://dx.doi.org/10.1074/jbc.ra120.012649.
Full textAbstract:
The gene encoding the proto-oncogene GTPase RAS is frequently mutated in human cancers. Mutated RAS proteins trigger antiapoptotic and cell-proliferative signals and lead to oncogenesis. However, RAS also induces apoptosis and senescence, which may contribute to the eradication of cells with RAS mutations. We previously reported that Ras association domain family member 6 (RASSF6) binds MDM2 and stabilizes the tumor suppressor p53 and that the active form of KRAS promotes the interaction between RASSF6 and MDM2. We also reported that Unc-119 lipid-binding chaperone (UNC119A), a chaperone of myristoylated proteins, interacts with RASSF6 and regulates RASSF6-mediated apoptosis. In this study, using several human cancer cell lines, quantitative RT-PCR, RNAi-based gene silencing, and immunoprecipitation/-fluorescence and cell biology assays, we report that UNC119A interacts with the active form of KRAS and that the C-terminal modification of KRAS is required for this interaction. We also noted that the hydrophobic pocket of UNC119A, which binds the myristoylated peptides, is not involved in the interaction. We observed that UNC119A promotes the binding of KRAS to RASSF6, enhances the interaction between RASSF6 and MDM2, and induces apoptosis. Conversely, UNC119A silencing promoted soft-agar colony formation, migration, and invasiveness in KRAS-mutated cancer cells. We conclude that UNC119A promotes KRAS-mediated p53-dependent apoptosis via RASSF6 and may play a tumor-suppressive role in cells with KRAS mutations.
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Wiechmann, Svenja, Pierre Maisonneuve, Britta M. Grebbin, Meike Hoffmeister, Manuel Kaulich, Hans Clevers, Krishnaraj Rajalingam, et al. "Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids." Journal of Biological Chemistry 295, no. 14 (February 20, 2020): 4526–40. http://dx.doi.org/10.1074/jbc.ra119.011025.
Full textAbstract:
The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.
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Evans, G. A., O. M. Z. Howard, R. Erwin, and W. L. Farrar. "Interleukin-2 induces tyrosine phosphorylation of the vav proto-oncogene product in human T cells: lack of requirement for the tyrosine kinase lck." Biochemical Journal 294, no. 2 (September 1, 1993): 339–42. http://dx.doi.org/10.1042/bj2940339.
Full textAbstract:
The haematopoietic protein, p95vav, has been shown to be a tyrosine kinase substrate and to have tyrosine kinase-modulated guanine-nucleotide-releasing-factor activity. This implies a function in the control of ras or ras-like proteins. Because ras activation has been shown to be a downstream event following stimulation of the interleukin-2 (IL-2) receptor, we investigated the possibility that vav was involved in IL-2 signal transduction pathways, using human T cells as a model. We found rapid tyrosine phosphorylation of vav in response to IL-2 within 1 min, with maximum increase of phosphorylation of 5-fold occurring by 5 min after treatment in normal human T cells. IL-2 stimulation of the human T-cell line YT and a subclone of the YT cell line (YTlck-) that does not express message for the src-family kinase p56lck also results in a rapid rate of tyrosine phosphorylation of vav of more than 5-fold by 5 min. These results suggest that vav may play an important role in IL-2-stimulated signal transduction and that there is not a strict requirement for the tyrosine kinase p56lck.