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Y Kaziro

Publications and source records attributed to Y Kaziro.

At least 55 records · Page 3Linked to original sources
PubMed1997

An activated mutant of R-Ras inhibits cell death caused by cytokine deprivation in BaF3 cells in the presence of IGF-I.

R-Ras belongs to a family of low molecular weight GTP-binding proteins and exhibits 55% amino acid identity to H-Ras. It has been demonstrated that H-Ras inhibits cell death caused by interleukin-3 (IL-3) withdrawal in BaF3 cells (Kinoshita et al. (1995b); Terada et al. (1995)). In the present study, we examined whether R-Ras also rescues BaF3 cells from the factor-deprived cell death. To do this, several BaF3 transfectants were established, in which expression of wild-type as well as mutant R-Ras was regulated by an inducible promoter. Using these transfectants, we found that expression of an activated R-Ras mutant, R-Ras (Q87L), suppressed the death of IL-3-deprived BaF3 cells. On the other hand, expression of the wild-type and the dominant-negative mutant of R-Ras showed no inhibitory effect on cell death, indicating that R-Ras x GTP abrogated cell death caused by deprivation of IL-3. Furthermore, it was found that IGF-I in serum was required for the anti-apoptotic activity of R-Ras. Suppression of cell death by R-Ras(Q87L) was inhibited by wortmannin, LY294002 (phosphatidylinositol 3-kinase (PI3K) inhibitors), or PD98059 (inhibitor for MEK, a specific activator of mitogen-activated protein kinase (MAPK)). In addition, we have shown that, in HEK293 cells, R-Ras and IGF-I could activate MAPK synergistically. Also, PI3K activity was co-immunoprecipitated with an activated mutant of R-Ras. These results suggest that R-Ras in collaboration with IGF-I suppressed apoptotic cell death of BaF3 caused by IL-3 deprivation, presumably by modulating the activitites of MAPK and PI3K.

Androstadienes↗
PubMed1997

Activation of Stat1 and subsequent transcription of inducible nitric oxide synthase gene in C6 glioma cells is independent of interferon-gamma-induced MAPK activation that is mediated by p21ras.

Rat C6 glioma cells have been used to characterize molecular events involved in the regulation of inducible nitric oxide synthase (iNOS) gene expression stimulated by interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). IFNs induce a signaling event which involves activation of Stat1 transcription factor. Previous studies have shown that IFNs also induce extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activation. However, the mechanisms by which IFNs stimulate MAPK activation remain elusive. Here we show that in C6 glioma cells, transiently expressing the dominant-negative form of c-Ha-Ras (Asn-17) abrogated IFN-gamma-induced ERK1 and ERK2 activation. Furthermore, PD98059, a specific MEK1 inhibitor, also blocked this activation. These results indicate that p21ras and MEK1 are required for IFN-gamma-induced ERK1 and ERK2 activation. Recent studies have reported that MAPK is responsible for serine phosphorylation of Stat1 which is required for Stat1's DNA binding and maximal transcriptional activity. Thus, we examined the role of the Ras-MAPK pathway in Stat1 activation and subsequent iNOS induction in C6 glioma cells. Further experiments showed that neither Asn-17 Ras expression nor concentrations of PD98059, which completely abrogated IFN-gamma-induced ERK1 and ERK2 activation, affected Stat1 DNA binding activity or iNOS induction, indicating that the Ras-MAPK pathway does not appear to be involved in the activation of Stat1 and subsequent iNOS induction in C6 glioma cells.

Animals↗
PubMed1997

C-terminal mutation of G protein beta subunit affects differentially extracellular signal-regulated kinase and c-Jun N-terminal kinase pathways in human embryonal kidney 293 cells.

G protein beta and gamma subunits (Gbeta and Ggamma) form a complex that is involved in various signaling pathways. We reported that the C-terminal 10 amino acids of Gbeta are required for association with Ggamma (Yamauchi, J., Kaziro, Y., and Itoh, H. (1995) Biochem. Biophys. Res. Commun., 214, 694-700). To evaluate further the significance of the C-terminal region of Gbeta in the formation of a Gbetagamma complex and its signal transduction, we constructed several C-terminal mutants and expressed them in human embryonal kidney 293 cells. The mutant lacking the C-terminal 2 amino acids (DeltaC2) failed to associate with Ggamma, whereas deletion of the C-terminal amino acid (DeltaC1), replacement of Trp at -2 position by Ala (W339A), and addition of six histidines ((His)6) at the C terminus did not affect the association with Ggamma. We also studied the effect of these mutations on the activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). Co-expression of the DeltaC2 or (His)6 mutant with Ggamma did not activate MAPK/ERK at all, whereas the DeltaC1 or W339A mutant showed the MAPK/ERK activation. The JNK/SAPK activity was stimulated by the W339A, DeltaC2, or (His)6 mutant, but not by the DeltaC1 mutant. These results suggest that the C-terminal region of Gbeta participates differentially in the signaling for MAPK/ERK and JNK/SAPK activations in mammalian cells.

Amino Acid Sequence↗
PubMed1997

Inducible high-level expression vector for mammalian cells, pEF-LAC carrying human elongation factor 1alpha promoter and lac operator.

We have constructed an inducible high-level expression vector, pEF-LAC. pEF-LAC has a modified human polypeptide chain elongation factor 1alpha (EF-1alpha) promoter containing three lactose operator sequences. Using the cat reporter gene, we characterized the transcriptional activity of pEF-LAC. In the transient transfection of NIH3T3 and BaF3 cells, the transcriptional activity of pEF-LAC was higher than that of the original human elongation factor 1alpha promoter, simian virus 40 (SV40) promoter, and Rous sarcoma virus (RSV) long terminal repeat (LTR). Cotransfection of the lactose repressor expression plasmid effectively suppressed the promoter activity of pEF-LAC, and the activity was fully recovered by addition of isopropyl beta-D-thiogalactopyranoside (IPTG). Even in the stable transfection of Rat-1 cells, the promoter activity of the integrated pEF-LAC was much higher than that of the RSV-LTR and regulated in an IPTG-dependent manner. These results suggest that pEF-LAC is a useful vector for the inducible high-level expression of the cloned gene in a variety of mammalian cells.

3T3 Cells↗
PubMed1997

Ras-dependent activation of c-Jun N-terminal kinase/stress-activated protein kinase in response to interleukin-3 stimulation in hematopoietic BaF3 cells.

Activation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase pathway in response to stimulation of the interleukin (IL)-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor was examined in mouse hematopoietic BaF3-derived cell lines (BaF3-N6 and -V2 cells). Significant increase in the activity of JNK1 was observed within 30 min following IL-3 or GM-CSF stimulation at physiological concentrations. Dominant-negative Ras(S17N), which is conditionally expressed in the presence of isopropyl-1-thio-beta-D-galactoside in BaF3-N6 cells, prevented the IL-3 stimulation of JNK1, whereas anisomycin-induced JNK1 activation was unaffected. Furthermore, a deletion mutant of the common beta subunit for IL-3 and GM-CSF receptors that consists of only the membrane-proximal region, including box 1 and box 2 motifs, was incapable of facilitating JNK1 activity as well as Ras activation. These results provide evidence that Ras is required for IL-3-stimulated JNK1 activation. We also examined if constitutively active Ras(G12V) alone could stimulate JNK1 activity by using the inducible expression system. Isopropyl-1-thio-beta-D-galactoside induction of Ras(G12V) in the BaF3-V2 cell line caused no significant increase in JNK1 activity, which could be activated by IL-3 or anisomycin. On the contrary, the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway was fully activated following Ras(G12V) induction. Together with these results, it seems likely that the Ras protein is indispensable for the IL-3 stimulation of JNK1 although Ras activation by itself is insufficient for JNK1 activation.

Animals↗
PubMed1996

Tyrosine phosphorylation of ACK in response to temperature shift-down, hyperosmotic shock, and epidermal growth factor stimulation.

The mammalian Cdc42 protein regulates various kinds of cellular responses, including formation of filopodia, polarization of T cells, and cell cycle progression. A non-receptor tyrosine kinase ACK, which specifically binds to the GTP-bound form of Cdc42, was isolated as a putative target of Cdc42. Here we show the induction of tyrosine phosphorylation of ACK in response to temperature shift-down to 25 degrees C, and hypertonic shock, as well as stimulation with epidermal growth factor (EGF) in human embryonic kidney (HEK) 293 cells. The increased tyrosine phosphorylation level upon temperature shift-down was sustained for at least 60 min, whereas reversion of the temperature to 37 degrees C caused rapid tyrosine dephosphorylation to the initial level. The responses to EGF and the high osmolarity were transient. Furthermore, we observed association of ACK with an adaptor protein Grb2, which may suggest the involvement of Grb2 in EGF receptor-mediated tyrosine phosphorylation of ACK.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Analysis of the T-cell activation signaling pathway mediated by tyrosine kinases, protein kinase C, and Ras protein, which is modulated by intracellular cyclic AMP.

T-cell receptor (TCR) triggering by an anti-CD3 antibody or phytohemagglutinin (PHA) as well as the treatment with phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), induces activation of Ras in T-lymphocytes (Downward, J. et al. (1990)) Nature 364, 719-723). In this paper, we studied the role of Ras in the process of TCR-mediated T-cell activation using a human lymphomic Jurkat cell line. The stimulatory effect of TCR cross-linking on Ras activation was inhibited by herbimycin A, a specific inhibitor of protein tyrosine kinases (PTKs), whereas PMA-induced Ras activation was not affected. On the other hand, calphostin C, a specific inhibitor of PKC, blocked not only PMA-induced, but also TCR-mediated formation of Ras.GTP. Furthermore, down-regulation of PMA-sensitive PKC severely impaired the activation of Ras in response to TCR-stimulation. Tyrosine-phosphorylation and translocation to the particulate fraction of phospholipase C-gamma 1 (PLC-gamma 1) were observed upon T-cell activation. Subcellular localization of PKC was also changed when the cells were stimulated with an anti-CD3 antibody or PMA. While TCR-stimulated translocation of PKC was observed only transiently, PMA-induced translocation of PKC was more sustained. These results suggest that the activation of PLC-gamma 1 by PTK and subsequent activation of PKC are important for TCR-mediated Ras activation in Jurkat cells. An activated form of Ras enhanced the activation of interleukin 2 (IL-2) promoter by TCR stimulation or PMA treatment, although the activated Ras by itself was insufficient for IL-2 promoter activation. On the other hand, a dominant-inhibitory Ras diminished almost completely the activation of IL-2 promoter induced by PMA plus calcium ionophore, indicating that Ras is essential for transduction of T-cell activation signals. Cholera toxin (CTX), which directly activates Gs alpha, is shown to inhibit the activation of IL-2 promoter. TCR-mediated Ras activation, tyrosine phosphorylation and translocation of cellular proteins including ZAP-70, PLC-gamma 1 , and PKC. An activated Gs alpha mutant as well as dibutylyl cAMP (dBcAMP) also showed similar inhibitory effects.

Cell Compartmentation↗
PubMed1996

Unfarnesylated transforming Ras mutant inhibits the Ras-signaling pathway by forming a stable Ras.Raf complex in the cytosol.

Farnesyltransferase inhibitors cause the growth arrest of ras-transformed cells, but not that of normal cells. To elucidate the mechanism of this differential effect, we examined the effect of accumulation of unfarnesylated Ras in the cytosol by using RasG12V,C186S and RasC186S, which mimic unfarnesylated form of the oncogenic and the normal Ras, respectively. We found that RasG12,C186S inhibited activation and membrane translocation of Raf by forming a stable complex with Raf in the cytosol. In contrast, RasC186S showed inhibitory effect on neither Raf activation nor Raf translocation. These results indicate that unfarnesylated oncogenic Ras interacts with Raf in the cytosol and inhibits its membrane translocation, a crucial step for the Raf activation, while unfarnesylated normal Ras does not.

Cell Line↗
PubMed1996

G protein specificity of the muscarine-induced increase in an inward rectifier potassium current in AtT-20 cells.

Muscarine and somatostatin enhance an inward rectifier K+ conductance in the AtT-20 pituitary cell line. Both effects are abolished by pertussis toxin (PTX). To determine which PTX-sensitive G protein mediates these agonist effects, we made cDNAs encoding mutant PTX-insensitive Gi alpha subtypes, in which the cysteine residue fourth from the C terminus was replaced with serine. The mutated cDNA was transfected into AtT-20 cells, resulting in stable cell lines overexpressing a Gi alpha subtype. As controls, wild-type Gi alpha cDNA was transfected into AtT-20 cells. The agonist-induced increase of the inward rectifier K+ conductance in the transfectants was examined with the whole-cell clamp method. Only in the cell lines into which the mutated (PTX-insensitive) Gi2 alpha cDNA was transfected, did the muscarine response become PTX-insensitive, suggesting that Gi2 couples to the muscarinic receptor and enhances the activity of the inward rectifier K+ channel. However, PTX-insensitive somatostatin responses were not obtained in any of the cell lines transfected with a mutated Gi alpha cDNA, suggesting either that none of the Gi subtypes is a transducer for the somatostatin effect or that the mutation prevents the coupling of the Gi alpha to the somatostatin receptor.

Animals↗
PubMed1995

Inhibition of Ras/Raf interaction by anti-oncogenic mutants of neurofibromin, the neurofibromatosis type 1 (NF1) gene product, in cell-free systems.

The neurofibromatosis type 1 (NF1) gene encodes a protein, neurofibromin, containing GTPase-activating protein-related domain (GRD) that stimulates intrinsic GTPase activity of Ras protein. By screening a randomly mutagenized NF1-GRD library in Saccharomyces cerevisiae, we isolated two NF1-GRD mutants (NF201 and NF204) with single amino acid substitutions, which suppress the heat shock-sensitive phenotype of the RAS2(G19V) mutant. The NF1-GRD mutants also suppress the oncogenic Ras-induced transformation of NIH 3T3 mouse fibroblasts (Nakafuku, M., Nagamine, M., Ohtoshi, A., Tanaka, K., Toh-e, A., and Kaziro, Y. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6706-6710). In this paper, we investigated the molecular mechanism of inhibition of the transforming Ras-specific function by the NF1-GRD mutants in mammalian cells. In human embryonic kidney (HEK) 293 cells, the mutant NF1-GRDs attenuated the stimulation of mitogen-activated protein kinase by Ras(G12V), but not by platelet-derived growth factor. In cell-free systems, purified recombinant NF1-GRD mutants showed an inhibitory effect on the association of Ras.guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with Raf at several times lower concentrations than the wild type. Furthermore, it was revealed that the binding affinity of the mutant NF1-GRDs toward Ras.GTP gamma S is approximately 5-10 times higher than the wild type. These results suggest that the mutant NF1-GRDs tightly bind to an oncogenic Ras in its GTP-bound active conformation and block the interaction between Ras and its effector, Raf.

3T3 Cells↗
PubMed1995

Ras is not required for the interleukin 3-induced proliferation of a mouse pro-B cell line, BaF3.

It has been demonstrated that Ras is involved in interleukin 3 (IL-3)-stimulated signal transduction in various hematopoietic cultured cells (Satoh, T., Nakafuku, M., Miyajima, A., and Kaziro, Y. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3314-3318; Duronio, V., Welham, M. J., Abraham, S., Dryden, P., and Schrader, J. W. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1587-1591). However, it has not been fully understood which of IL-3-promoted cellular responses, i.e. proliferation, survival, and differentiation, requires Ras function. We employed a system of inducible expression of the dominant-negative (S17N) or dominant-active (G12V) mutant of Ras in BaF3 mouse pro-B cell line to analyze the role of Ras in IL-3-stimulated signal transduction. Induction of the dominant-negative Ras(S17N) effectively inhibited the IL-3-induced activation of c-Raf-1 and mitogen-activated protein kinase (MAPK). Furthermore, the activation of fos gene promoter following IL-3 stimulation was almost completely abolished when Ras(S17N) was induced. Under these conditions, Ras(S17N) exhibited no inhibitory effect on IL-3-dependent proliferation assessed by the increase of cell numbers and a mitochondrial enzyme activity. The results indicate that Ras-dependent pathways, including the Raf/MAPK/Fos pathway, are dispensable for IL-3-induced growth stimulation. When BaF3 cells were treated with a tyrosine kinase inhibitor, herbimycin A, IL-3-dependent proliferation of the cells was impaired, suggesting that tyrosine kinase-mediated pathways are critical for growth promotion. On the other hand, apoptotic cell death caused by deprivation of IL-3 was prevented by the induction of the activated mutant Ras(G12V), although the rate of cell number increase was markedly reduced. Thus, it is likely that Ras-independent pathways play important roles to facilitate the proliferation although they may not be essential for IL-3-stimulated antiapoptotic signal transduction.

Animals↗
PubMed1995

Carboxyl terminal of G protein beta subunit is required for association with gamma subunit.

To investigate a complex formation of heterotrimeric G protein beta and gamma subunits, we constructed a deletion mutant which lacked the C-terminal WD40 repeating unit of beta 1 and cotransfected into HEK293 cells with gamma 2 which was tagged with FLAG-epitope. Co-immunoprecipitation assay indicated that the mutant beta 1 failed to associate with FLAG gamma 2. Furthermore, from the analysis of additional deletion mutants, the last 10 amino acids of C-terminal region of beta 1 were found to be necessary to associate with gamma 2. These results suggest that the C-terminal region, in addition to the N-terminal alpha-helical structure, of beta subunit may be involved in the beta gamma complex formation.

Amino Acid Sequence↗
PubMed1995

G protein beta gamma subunit activates Ras, Raf, and MAP kinase in HEK 293 cells.

Using transient transfection of HEK 293 cells, we have studied the activation of Ras, c-Raf, and MAP kinase by G protein-coupled receptors, activated G protein alpha subunit (G alpha), and beta gamma subunits (G beta gamma). The expression of constitutively activated Gs alpha, Gi alpha, and G11 alpha did not have any effect on MAP kinase phosphorylation. In contrast, overexpression of G beta gamma could stimulate the phosphorylation of MAP kinase and enhance the MEK kinase activity of c-Raf. Coexpression of dominant negative Ras inhibited G beta gamma-induced phosphorylation of MAP kinase. Furthermore, the GTP-bound form of Ras was increased by overexpression of G beta gamma. These results strongly suggest that the G beta gamma may play an important role in signaling from G protein-coupled receptors to the MAP kinase pathway, and the activation of Ras and c-Raf may be involved in this signaling cascade in HEK 293 cells.

Cell Line↗
PubMed1994

Signal transduction pathways from insulin receptors to Ras. Analysis by mutant insulin receptors.

We have examined the involvement of insulin receptor (IR) substrate-1 (IRS-1) and/or Shc in the upstream of Ras activation in insulin signaling using Chinese hamster ovary (CHO) cell lines overexpressing wild-type (CHO-IR) cells) or mutant insulin receptors. In CHO-IR cells, insulin rapidly phosphorylated IRS-1 and Shc at tyrosine residues and stimulated the formation of the active GTP-bound Ras (Ras.GTP). In contrast, a CHO cell line overexpressing the kinase-negative mutant insulin receptor substituting Arg1018 for Lys1018 was unable to tyrosine-phosphorylate IRS-1 and Shc and failed to activate Ras in response to insulin. A CHO cell line overexpressing the mutant insulin receptor, substituting Ala960 for Tyr960 and which was known to exhibit impaired tyrosine phosphorylation of IRS-1 and biological effects evoked by insulin, showed severely impaired insulin-dependent tyrosine phosphorylation of Shc and moderately impaired activation of Ras. Another cell line overexpressing the mutant insulin receptor, lacking 82 amino acids of the C terminus of beta-subunit and which was recently reported to retain normal insulin-dependent tyrosine phosphorylation of IRS-1, showed slightly impaired Ras activation at 10(-7) M insulin with severely reduced tyrosine phosphorylation of Shc protein. Furthermore, insulin did not induce the association of tyrosine-phosphorylated IRS-1 and Shc in CHO-IR cells. These results suggest that Shc and IRS-1 lie in the separate signaling pathways and that the tyrosine phosphorylation of IRS-1 with or without some low level of Shc phosphorylation may be enough to stimulate the submaximal accumulation of Ras.GTP complex and may need synergistically the higher level of tyrosine phosphorylation of Shc to induce the full activation of Ras in insulin signaling.

Animals↗
PubMed1994

Mutations that abolish the ability of Ha-Ras to associate with Raf-1.

Recent studies have revealed that Ras can associate physically with Raf. In the present study, we tested 34 mutants of Ha-Ras carrying substitution(s) in the region of residues 23-71 for their ability to associate with Raf-1. Mouse Ba/F3 cell lysates were incubated with each mutant Ras protein, in either the guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)- or the guanosine 5'-[beta-thio]diphosphate (GDP beta S)-bound form, and the anti-Ras antibody Y13-238. The immunoprecipitates were analysed for the presence of Raf-1 by Western blotting with an anti-Raf-1 antibody. Six mutants of Ras, E31K, P34G, T35S, D38N, D57A and A59T, failed to bind Raf-1. Mutations N26G, V29A, S39A, Y40W, R41A, V44A, V45E, L56A and T58A partially reduced the ability to bind Raf-1. All the other mutants could associate with Raf-1 with nearly the same efficiency as that of wild-type Ras. Thus, the Raf-I-binding ability of Ras appears to be affected by mutations in the N-terminal region, and in particular, by those in and neighboring the effector region (residues 32-40) and in the region (residues 56-59) flanking the N-terminal of Switch II. The abilities to bind Raf-1 and to induce neurite outgrowth of pheochromocytoma (PC) 12 cells correlate to each other for 22 Ras mutants. However, mutation A59T, which does not reduce the neurite-inducing or transforming activities, abolishes the ability to bind Raf-1. In contrast, mutations Y32F, K42A and L53A, which impair the neurite-inducing activity of Ras, have no effect on the Ras.Raf-1 association. Partially reduced Raf-1-binding ability was observed for mutants V29A, S39A, Y40W, R41A, V44A, L56A and T58A, which exhibit full neurite-inducing activity, and also for mutant V45E, which has no activity of neurite induction.

Animals↗
PubMed1993

Sequence of the cDNA encoding Ras GTPase-activating protein from rat.

We cloned and sequenced a 3296-bp cDNA encoding the rat Ras GTPase-activating protein (GAP). Comparison of the nucleotide (nt) and deduced amino acid (aa) sequences to those of previously described GAP cDNAs revealed greater than 90% identity. Homology is highest between rat and mouse GAP both at the nt (96% identity) and deduced aa levels (98% identity).

Amino Acid Sequence↗
PubMed1993

GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells.

Ras is involved in signal transduction of various factors for growth, differentiation, and oncogenesis. Recent studies have revealed several proteins that function upstream and downstream of the Ras signaling pathway. However, its immediate downstream target molecular has not yet been identified. In an effort to identify the Ras-associated downstream proteins, we added recombinant Ha-Ras in a GTP-bound form to cell-free lysates and used several antibodies against Ras to immunoprecipitate Ras complexes. We found that a serine/threonine kinase, Raf-1, was coimmunoprecipitated with Ha-Ras by two anti-Ras antibodies (LA069 and Y13-238), whereas a neutralizing antibody against Ras (Y13-259) could not precipitate Raf-1. The coimmunoprecipitation was observed with a complex of Ras and guanosine 5'-[gamma- thio]triphosphate but not with a complex of Ras and guanosine 5'-[beta-thio]diphosphate. The GTP-dependent association of Ha-Ras with Raf-1 was observed with lysates of various types of cultured cells, including NIH 3T3, pheochromocytoma (PC) 12, Ba/F3, and Jurkat T cells, and also with crude extracts from rat brain. Furthermore, Raf-1 was precipitated with a transforming Ha-Ras mutant ([Val12]Ras) and wild-type Ha-Ras but not with an effector-region mutant ([Leu35,ARg37]Ras) that lacks transforming activity. These results indicate that Ras.GTP physically associates with Raf either directly or through other component(s) and strongly suggest that Raf functions in close downstream proximity to Ras in mammalian cells.

3T3 Cells↗