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Biomedical subjects

Y Kaziro

Publications and source records attributed to Y Kaziro.

At least 73 records · Page 4Linked to original sources
PubMed1993

Suppression of oncogenic Ras by mutant neurofibromatosis type 1 genes with single amino acid substitutions.

NF1 was first identified as the gene responsible for the pathogenesis of the human genetic disorder neurofibromatosis type 1. cDNA cloning revealed that its putative protein product has a domain showing significant sequence homology with the mammalian Ras GTPase activating protein and two yeast Saccharomyces cerevisiae proteins, Ira1 and Ira2. The Ras GTPase activating protein-related domain of the NF1 gene product (NF1-GRD) stimulates GTPase activity of normal Ras proteins but not of oncogenic mutant Ras from both mammalian and yeast cells. Thus, in yeast, NF1-GRD can suppress the heat-shock-sensitive phenotype of ira- cells but not the same phenotype of activated RAS such as RAS2Val19 and RAS2Leu68. We have screened a pool of mutagenized NF1 expression plasmids and obtained two mutant NF1 cDNA clones that can suppress the heat-shock-sensitive phenotype of RAS2Val19 cells. One clone (NF201) suppressed RAS2Leu68, RAS2Ser41, and RAS2Val19, whereas another clone (NF204) preferentially suppressed RAS2Val19. When expressed in mammalian cells, these mutant NF1-GRDs were able to induce the morphological reversion of v-ras-transformed NIH 3T3 cells. Both wild-type and mutant NF1-GRDs can stimulate the GTPase activity of normal but not transforming Ras. We suggest that mutant NF1-GRDs may bind tightly to transforming Ras, which stays in GTP-bound conformation, thus preventing the interaction with the putative effector molecule. On the other hand, normal Ras cannot be sequestered since the bound GTP is rapidly hydrolyzed upon interaction with mutant NF1-GRD to yield Ras-GDP, which is readily released from the NF1-GRD and recycled.

3T3 Cells↗
PubMed1993

Epidermal growth factor and transforming growth factor-alpha can induce neuronal differentiation of rat pheochromocytoma PC12 cells under particular culture conditions.

In rat pheochromocytoma PC12 cells, NGF induces neuronal differentiation. Upon stimulation with NGF, Ras is activated to a GTP-bound form, and the activated Ras can induce neuronal differentiation. Recently, we and others observed that epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) can also activate Ras in PC12 cells. This is puzzling since previous reports indicated that EGF stimulates proliferation rather than differentiation in PC12 cells. In this paper, we re-examined the biological effect of EGF and TGF-alpha, and found that these factors can also induce neuronal differentiation under particular culture conditions. Not only the outgrowth of long neurites, but the induction of neurofilament proteins and the metalloprotease transin was also observed in the EGF- and TGF-alpha-stimulated cells. These data clearly indicate that in addition to NGF, EGF and TGF-alpha can also induce the differentiation of PC12 cells under particular conditions.

Animals↗
PubMed1993

A mutant substituting valine for glycine at position 49 of Gs alpha induces neuronal differentiation of PC12 cells without activation of adenylate cyclase.

A GTPase-deficient mutant of the alpha-subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylate cyclase, substituting valine for glycine 49 (G49V) was transiently expressed in COS7 cells. The basal level of cAMP as well as an agonist-dependent accumulation of cAMP was two-fold higher in transfectants of Gs alpha (G49V) than in those of the normal counterpart. A stable transformant of PC12 cells expressing Gs alpha (G49V) under the control of a metallothionein promoter was then established. Two independent clones showed neurite outgrowth when the mutated Gs alpha was expressed by adding Cd2+ to culture medium. However, the level of basal cAMP of the transformant of PC12 cells with Gs alpha (G49V) was lower than that of the parental cells. The response to an agonist of the adenosine A2-receptor was suppressed in transformants. Although a cAMP-responsive element (CRE) was slightly activated by transfection and transient expression of Gs alpha in PC12 cells, no activation but a suppression of CRE was observed with PC12 cells transiently expressing Gs alpha (G49V). These results suggest that the mutant Gs alpha (G49V) couples adenylate cyclase in a different way in PC12 cells and may transduce differentiation signals through a cAMP-independent pathway.

Adenylyl Cyclases↗
PubMed1993

Analysis of the expression of seven G protein alpha subunit genes in hematopoietic cells.

Various heterotrimeric GTP-binding proteins may have important functions in hematopoietic cells. There has been no comprehensive information, however, regarding their expression in various-lineage hematopoietic cells. In this report, the expression level of seven G protein alpha subunits (Gs alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Go alpha-1, Go alpha-2, and Gx alpha) in 13 hematopoietic cell lines were analyzed by Northern blot analysis. Gi1 alpha, Go alpha-1, Go alpha-2, and Gx alpha, were expressed in a limited number of cell lines whereas Gs alpha, Gi2 alpha, and Gi3 alpha were expressed ubiquitously in nearly all cell lines tested. Gi1 alpha was expressed selectively in a pre-T cell line, P30/PHK among lymphoid-lineage cell lines and a myeloblastic cell line, KG-1 among myelomonocytoid cell lines. Go alpha-1 was expressed only in a chronic myelocytic-leukemia cell line, K-562, whereas Go alpha-2 was not expressed in any cell lines tested after ordinary exposure of autoradiography (within 4 days). Gx alpha was expressed abundantly in a rat basophilic-leukemia cell line, RBL-2H3, and expressed in K-562. A barely detectable amount of Gx alpha messenger ribonucleic acid (mRNA) was found after a long exposure of autoradiography in several cell lines with megakaryoblastoid phenotype.

Animals↗
PubMed1993

Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types.

A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells. Therefore, it is likely that several different PDGF receptor-mediated signaling pathways function upstream of Ras, and the extent of the contribution of each pathway for the regulation of Ras may differ among different cell types.

Animals↗
PubMed1992

Interleukin 2-induced activation of Ras requires two domains of interleukin 2 receptor beta subunit, the essential region for growth stimulation and Lck-binding domain.

Interleukin 2 (IL-2) can stimulate the proliferation of various kinds of T-cell lines. The receptor for IL-2 is composed of at least two subunits (alpha and beta), of which beta subunit plays the major role in transducing growth signals into the cells. A nonreceptor-type tyrosine kinase, Lck, is associated with IL-2 receptor beta subunit, and the binding of IL-2 to its receptor induces the activation of Lck. On the other hand, it has been shown that stimulation of T-cells with IL-2 causes rapid activation of Ras protein. In this paper, we describe that both of the two regions in IL-2 receptor beta subunit, the indispensable region for the induction of cell growth (serine-rich region) and the binding region of Lck protein (acidic region), are required for the activation of Ras. These two regions are also required for tyrosine phosphorylation of an 85-kDa cellular protein (p85) and the accumulation of fos and jun mRNAs. This observation suggests also that the activation of a receptor-associated tyrosine kinase in response to IL-2-stimulation is primarily responsible for subsequent activation of the pathway through Ras to Fos and Jun.

Animals↗
PubMed1992

Differentiation factors, including nerve growth factor, fibroblast growth factor, and interleukin-6, induce an accumulation of an active Ras.GTP complex in rat pheochromocytoma PC12 cells.

Ras has been thought to be involved in neuronal differentiation of rat pheochromocytoma PC12 cells. PC12 cells are immature adrenal chromaffin-like cells which undergo differentiation to sympathetic neuron-like cells in response to nerve growth factor (NGF). Fibroblast growth factor (FGF) and interleukin (IL)-6 can also induce differentiation of PC12 cells. In this paper, we report that NGF, FGF, and IL-6 induce an accumulation of an active Ras.GTP complex. In the serum-starved culture of PC12 cells, 6% of the Ras protein was complexed with GTP. Upon stimulation with NGF, the percentage of Ras.GTP increased to 24% after 2 min, and the high level of Ras.GTP was maintained for at least 16 h. On the other hand, the activation of Ras by FGF and IL-6 showed distinct kinetics; about 3-fold increase of Ras.GTP was detected at 10 min, and afterward, the level returned to the basal level within 60 min. These observations provide direct evidence that activation of Ras is involved in signal transduction from these differentiation factors. In addition, it was found that growth factors, including epidermal growth factor, insulin, and insulin-like growth factor-I, and a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), can also activate Ras under the same conditions. A tyrosine kinase-specific inhibitor, genistein, inhibited the increase of Ras.GTP induced by NGF and other factors. On the other hand, down-regulation of protein kinase C (PKC) by prolonged treatment with TPA, which sufficiently blocked TPA-induced Ras activation, did not abolish the formation of Ras.GTP by NGF. These results suggest that tyrosine kinases rather than PKC play a major role in the NGF-induced activation of Ras in PC12 cells.

Animals↗
PubMed1992

Characterization and site-directed mutagenesis of a low M(r) GTP-binding protein, ram p25, expressed in Escherichia coli.

The ram gene encodes a GTP-binding protein with a M(r) of 25,068 (Nagata, K., Satoh, T., Itoh, H., Kozasa, T., Okano, Y., Doi, T., Kaziro, Y., and Nozawa, Y. (1990) FEBS Lett. 275, 29-32). It has a putative effector domain very similar to that of yeast SEC4 protein, and shares 40% identity and 60% homology with it, respectively. In order to analyze the biochemical properties, ram cDNA was engineered and inserted into a bacterial expression vector; this allowed the production at a high level of soluble recombinant ram p25 in Escherichia coli. The purified ram p25 contained an equimolar amount of GDP. The purified protein bound approximately 1 mol of [35S]guanosine 5'-O-(thiotriphosphate) GTP gamma S)/mol of protein, with a Kd value of 120 nM. [35S]GTP gamma S binding to this protein was inhibited by GTP and GDP, but not by ATP and ADP. In the presence of 10 mM Mg2+, the dissociation of [8,5'-3H]GDP and [35S]GTP gamma S from ram p25 occurred with rates of 0.015 min-1 and 0.004 min-1, respectively, showing that the ram p25 has a higher affinity for GTP than GDP. The rate of release of Pi from [gamma-32P]GTP-bound ram p25 was calculated to be 0.011 min-1. The contribution of guanine nucleotide-binding and GTP-hydrolysis domains of the protein to its biochemical activities was investigated by site-directed mutagenesis. Substitution of Val for Gly at position 19 resulted in disappearance of [35S]GTP gamma S- and [3H]GDP-binding activity in spite of good expression of the protein. Mutations of Thr41 to Ser, Ala76 to Thr, and Asn133 to His slightly increased the rates of [35S] GTP gamma S binding and [3H]GDP dissociation, but had almost no effects on the manner of [gamma-32P]GTP hydrolysis. Replacement of Gln78 with Leu significantly increased the [3H]GDP dissociation rate (7-fold) and decreased GTP hydrolytic activity considerably.

Amino Acid Sequence↗
PubMed1992

Inhibition of interleukin 3 and granulocyte-macrophage colony-stimulating factor stimulated increase of active ras.GTP by herbimycin A, a specific inhibitor of tyrosine kinases.

Interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate the proliferation of several kinds of cultured hematopoietic cell lines. Growth signals from IL-3 and GM-CSF cause accumulation of active Ras.GTP complexes in PT18 mouse mast cell line (Satoh, T., Nakafuku, M., Miyajima, A., and Kaziro, Y. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3314-3318). In this paper we describe the effect of herbimycin A, a tyrosine kinase-specific inhibitor, on the activation of Ras. The increase of Ras.GTP induced by IL-3 and GM-CSF diminished in cells treated with 0.5 approximately 1 micrograms/ml of herbimycin A for 24 h prior to the addition of the growth factors. Under this condition, the extent of phosphorylation on tyrosine residues of proteins decreased. However, the activity of cAMP-dependent protein kinase and protein kinase C did not change. Growth of cells in the presence of IL-3 or GM-CSF was also completely inhibited. These observations suggest that tyrosine kinases are involved in the pathways between IL-3 and GM-CSF receptors and Ras and that they are essential for the growth stimulated by these growth factors.

Animals↗
PubMed1992

Ras in signal transduction.

Ras protein is a GTP-binding protein, and acts as a signal transducer in fibroblast, lymphoid, myeloid, and neuronal cells. In all cases, tyrosine kinases, intrinsic to or associated with receptors, seem to play an important role for the activation of Ras in response to extracellular stimulations. A GDP/GTP exchange regulator and a GTPase stimulatory protein are thought to mediate signals from the kinases. The active Ras.GTP can cause different phenotypes, that is, proliferation, transformation, activation, or differentiation, depending on cell types, although it is not yet clear what is the primary target of the active Ras-GTP or how the various phenotypes are determined downstream of Ras protein.

Animals↗
PubMed1991

Interactions of bovine mitochondrial phenylalanyl-tRNA with ribosomes and elongation factors from mitochondria and bacteria.

A homologous in vitro poly(U)-directed translation system has been established using animal mitochondrial ribosomes, elongation factors (EF) and phenylalanyl-tRNA(Phe). The rate of incorporation of phenylalanine into polyphenylalanine in the mitochondrial system is slower than that observed for the homologous Escherichia coli system. E. coli ribosomes can be used in place of mitochondrial ribosomes in this system with only a slight decrease in the efficiency of phenylalanine incorporation from mitochondrial Phe-tRNA. However, E. coli elongation factor Tu (EF-Tu) cannot replace the mitochondrial EF-Tu in promoting the use of mitochondrial Phe-tRNA. The interaction between EF-Tu and mitochondrial Phe-tRNA was investigated by using the ability of EF-Tu to protect the aminoacyl-tRNA bond from hydrolysis. These results showed that both mitochondrial and E. coli EF-Tus are capable of interacting with mitochondrial Phe-tRNA. However, ribosomal A-site binding assays demonstrated that efficient binding of the mitochondrial Phe-tRNA to the ribosomal A-site was only obtained with the homologous mitochondrial EF-Tu.

Animals↗
PubMed1991

Analysis by mRNA levels of the expression of six G protein alpha-subunit genes in mammalian cells and tissues.

The distribution and levels of expression of Gs alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Go alpha, and Gx alpha mRNAs were compared by Northern blot analysis using several rat tissues and selected human and rat cell lines. Gi1 alpha, Go alpha, and Gx alpha, were detected in a limited number of tissue and cells whereas Gi2 alpha, Gi3 alpha, and Gs alpha, were expressed in all the tissues and cells tested albeit in varying amounts. The expression of these six genes appears to be differentially regulated during postnatal development of the rat brain. High expression levels particularly of Go alpha, in young rat brain may be related to the formation of neurites during differentiation of nerve cells.

Age Factors↗
PubMed1991

Amino acid sequence determination of the novel forms of Go alpha purified from bovine brain membranes.

Previously we have reported that there are at least four different forms of Go alpha in bovine brain membranes which can be distinguished by their elution profiles from Mono Q column and their immunological reactivities. The four alpha-subunits are referred to as alpha o1, alpha o2, alpha o3 and alpha o4 in their elution orders from the column. Partial amino acid sequences of the purified alpha o1 and alpha o2 were determined and compared with the predicted sequences of two classes of Go alpha cDNAs, termed Go alpha-1 and Go alpha-2. There were at least two unique fragments corresponding with the predicted amino acid sequence of the Go alpha-2 cDNA but different from that of the Go alpha-1 cDNA upon tryptic digestion of alpha o1- or alpha o2-subunit. The alpha o3- and alpha o4-subunits, but not alpha o1-and alpha o2-subunits, were recognized by an antibody raised against a unique amino acid sequence predicted from Go alpha-1 cDNA. These results suggest that alpha o1,2 subunits and alpha o3,4 subunits are encoded by Go alpha-2 cDNA and Go alpha-1 cDNA, respectively.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of a gene encoding a G-protein alpha subunit from Schizosaccharomyces pombe: involvement in mating and sporulation pathways.

The gpal gene of Schizosaccharomyces pombe, which encodes a protein homologous with the alpha subunits of mammalian guanine nucleotide-binding proteins (G proteins), was isolated by cross-hybridization using rat Gi1 alpha and Gx alpha cDNA. The deduced amino acid sequence was about 37% identical with rat Gi1 alpha and Gx alpha proteins and contained three conserved motifs commonly found in all GTP-binding proteins. Disruption of gpa1 was not lethal but conferred sterility and sporulation deficiency on Sch. pombe cells. Thus, the gene is essential for the sexual development and is probably coupled to mating-factor receptors. In contrast to Saccharomyces cerevisiae GPA1, which plays a negative role in mating-factor signal transduction, Sch. pombe gpa1+ apparently has a positive function. A gpa1 transcript of 2.2 kilobases was detected in vegetatively growing cells. A 1.6-kilobase gpa1 transcript appeared in addition to the 2.2-kilobase transcript when cells were derepressed for mating or meiosis.

Amino Acid Sequence↗
PubMed1991

Structure of the human gene and two rat cDNAs encoding the alpha chain of GTP-binding regulatory protein Go: two different mRNAs are generated by alternative splicing.

Go is a specific class ("other") of signal-transducing heterotrimeric GTP-binding proteins (G proteins) that is expressed in high levels in mammalian brain. We have cloned two different rat cDNAs encoding the alpha subunit of Go (Go alpha-1 and Go alpha-2) and a human Go alpha chromosomal gene. The human Go alpha gene spans more than 100 kilobases and contains 11 exons, including one noncoding exon in the 3' flanking region. The 5' flanking region is highly G + C-rich and contains five G.C boxes (Sp1 binding sites) but no TATA box. Exons 7 and 8 coding for amino acid residues 242-354 of Go alpha protein are duplicated (referred to as exons 7A, 7B, 8A, and 8B). It was found that exons 7A and 8A code for Go alpha-1, and 7B and 8B code for Go alpha-2. This indicates that two different Go alpha mRNAs may be generated by alternative splicing of a single Go alpha gene. The splice sites of the Go alpha-1 and Go alpha-2 genes are completely identical with those encoding human inhibitory G protein alpha subunits Gi2 alpha and Gi3 alpha [Itoh, H., Toyama, R., Kozasa, T., Tsukamoto, T., Matsuoka, M. & Kaziro, Y. (1988) J. Biol. Chem. 263, 6656-6664] and also transducin G protein alpha subunit Gt1 alpha [Raport, C. J., Dere, B. & Hurley, J. (1989) J. Biol. Chem. 264, 7122-7128]. Sequence homology and conservation of the exon-intron organization indicate that the genes coding for Go alpha, Gi2 alpha, Gi3 alpha, Gt1 alpha, and probably Gi1 alpha may be evolved from a common progenitor. Like Go alpha-1, Go alpha-2 is expressed mainly in brain.

Amino Acid Sequence↗
PubMed1991

Involvement of ras p21 protein in signal-transduction pathways from interleukin 2, interleukin 3, and granulocyte/macrophage colony-stimulating factor, but not from interleukin 4.

The protooncogene ras acts as a component of signal-transduction networks in many kinds of cells. The ras gene product (p21) is a GTP-binding protein, and the activity of the protein is regulated by bound GDP/GTP. Recent studies have shown that a certain class of growth factors stimulates the formation of active p21-GTP complexes in fibroblasts and that oncogene products with enhanced tyrosine kinase activities have a similar effect on ras p21. We have measured the ratio of active GTP-bound p21 to total p21 in several lymphoid and myeloid cell lines in order to understand the role of ras in the proliferation of these cells. Interleukin 2 (IL-2), IL-3, and granulocyte/macrophage colony-stimulating factor (GM-CSF) enhance the formation of the active p21.GTP, whereas IL-4 has no effect on p21-bound GDP/GTP. These results strongly suggest that ras p21 acts as a transducer of signals from IL-2, IL-3, and GM-CSF, but not from IL-4.

Animals↗
PubMed1991

Molecular cloning and expression of a cDNA encoding the secretin receptor.

Secretin is a 27 amino acid peptide which stimulates the secretion of bicarbonate, enzymes and potassium ion from the pancreas. A complementary DNA encoding the rat secretin receptor was isolated from a CDM8 expression library of NG108-15 cell line. The secretin receptor expressed in COS cells could specifically bind the iodinated secretin with high and low affinities. Co-expression of the secretin receptor with the alpha-subunit of rat Gs protein increased the concentration of the high affinity receptor in the membrane fraction of the transfected COS cells. Secretin could stimulate accumulation of cAMP in COS cells expressing the cloned secretin receptor. The nucleotide sequence analysis of the cDNA has revealed that the secretin receptor consists of 449 amino acids with a calculated Mr of 48,696. The secretin receptor contains seven putative transmembrane segments, and belongs to a family of the G protein-coupled receptor. However, the amino acid sequence of the secretin receptor has no significant similarity with that of other G protein-coupled receptors. A 2.5 kb mRNA coding for the secretin receptor could be detected in NG108-15 cells, and rat heart, stomach and pancreatic tissue.

Amino Acid Sequence↗