DOCSLIB.ORG

Regulation of Smooth Muscle A-Actin Promoter in Ras-Transformed Cells: Usefulness for Setting up Reporter Gene-Based Assay System for Drug Screening

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Regulation of Smooth Muscle A-Actin Promoter in Ras-Transformed Cells: Usefulness for Setting up Reporter Gene-Based Assay System for Drug Screening [CANCER RESEARCH 52, 6877-6884, December 15, 1992] Regulation of Smooth Muscle a-Actin Promoter in ras-transformed Cells: Usefulness for Setting Up Reporter Gene-based Assay System for Drug Screening C. Chandra Kumar, ~ Pierre Bushel, Sheela Mohan-Peterson, 2 and Fernando Ramirez Department of Tumor Biology, Schering-Plough Research Institute, Bloomfield, New Jersey 07003 ABSTRACT cancer have impaired GTPase activity, are insensitive to GAP, and remain bound to GTP leading to uncontrolled cell growth Oncogenic activation of ras results in changes in the transcription of (6). In addition, a number of posttranslational modifications of several genes leading to uncontrolled cell growth. In this paper, we ras such as palmitoylation, farnesylation, proteolytic cleavage, demonstrate that transformation of fibroblast cells by the ras oncogene leads to transcriptional repression of the smooth muscle a-actin pro- and carboxymethylation have been found to be essential for moter. Transient transfection analysis of plasmids containing the 5' biological activity of ras (9-11). upstream region of the human a-actin gene fused to human growth A number of approaches are being taken to find drugs that hormone or bacterial chloramphenicol acetyltransferase coding se- inhibit the accumulation of the GTP-bound form of the ras p21 quences into Rat-2 and ras-transformed Rat-2 (HO6) cells indicates that protein. These include assay systems designed to identify drugs a-actin promoter is repressed in ras-transformed cells. In addition, that inhibit the nucleotide exchange activity of ras, or stimulate stable rat fibroblast cell lines expressing human growth hormone or the GTPase activity of mutant ras, or inhibit ras:GAP interac- B-galactosidase under the control of ~,-actin promoter exhibit repressed tion, etc. (11). Recently, the enzymes involved in the modifica- reporter gene activity following transformation by the ras oncogene. tion of ras proteins have also been targeted for new drug dis- a-Actin promoter-driven B-galactosidase activity is derepressed in re- vertants of ras-transformed stable cell lines. This revertant cell line covery (11). Most of these assay systems are in vitro based, and expresses elevated levels of ras p21 protein and is resistant to retrans- the lead compounds in these assay systems have to be evaluated formation by Ki and Ha-ras oncogenes. The revertant may have either in secondary cell-based assay systems to determine their effi- a defective target protein whose activity is essential for the transforming cacy. In this paper, we describe a general cell-based assay sys- activity of ras or an activated tumor suppressor gene which can suppress tem capable of detecting compounds that inhibit the transform- the activity of ras. These results indicate that smooth muscle a-actin ing activity of ras, either directly or indirectly. promoter activity is a sensitive marker to follow phenotypic changes Previous studies have shown that human sm forms of myo- following transformation by ras and subsequent reversion. The advan- sin light chain 2 (MLC-2) and a-actin mRNA and protein tages of this a-actin promoter-reporter gene assay system to screen for levels are repressed in ras-transformed fibroblast cells (12, 13). drugs that inhibit the transforming activity of ras, either directly or These changes are part of the cytoskeletal changes that occur indirectly, are discussed. following neoplastic transformation of fibroblast cells (14). We have also shown that revertants of ras-transformed cells show INTRODUCTION normal levels of MLC-2 gene expression, suggesting that the expression of these cytoskeletal markers, such as sm MLC-2 ras proteins play a central role in neoplasia and growth con- and sm a-actin, is modulated by the ras-transformed pheno- trol. Activated ras oncogenes have been implicated in the onset type (12). In this paper, using plasmids containing 5' upstream of 20% of human cancers and greater than 90% of pancreatic sequences of the human a-actin gene fused to different reporter and colon carcinomas (1-3). Hence, drugs that interfere with genes, we first demonstrate that a 906-base pair fragment is the function of ras, either directly or indirectly, will be useful in the treatment of a number of cancers. A great deal of effort has sufficient and necessary to confer responsiveness to the ras oncogene. Using stable rat fibroblast cell lines that express been focused in understanding the biochemical and structural characteristics of the ras p21 protein (4, 5). The mechanisms by LacZ reporter gene activity from the a-actin promoter, we which ras can induce transformation of cells have also been further show that revertants of ras-transformed cells exhibit studied in great detail (6). ras p21 protein exhibits GTPase derepression of promoter activity. The revertant cell line ex- activity and exists either in GTP-bound activated form or in presses elevated levels of the ras p21 protein and is resistant GDP-bound inactive form. Proteins that modulate the activity to retransformation by Ki and Ha-ras oncogenes. The useful- of ras consist of (a) a nucleotide exchange factor that facilitates ness of this promoter system to identify agents that can inhibit the exchange of GTP/GDP and thus promotes the activation of the transforming activity of ras, either directly or indirectly, is ras (7) and (b) a protein that acts as a GAP 3 and promotes discussed. inactivation of ras (8). Mutant forms of ras found in human MATERIALS AND METHODS Received 6/19/92; accepted 9/30/92. Cell Culture and Transfections The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accord- Rat-2 fibroblast cells obtained from the American Type Culture Col- ance with 18 U.S.C. Section 1734 solely to indicate this fact. lection (CRL 1764) and stably transfected derivatives were grown in 1To whom requests for reprints should be addressed. 2 Present address: DNAX Research Institute, Palo Alto, CA 94304. DMEM supplemented with 2 mM glutamine and 10% (vol/vol) fetal 3 The abbreviations used are: GAP, GTPase-activating protein; sm, smooth bovine serum. Rat-6 fibroblasts, obtained from Dr. Bernard Weinstein muscle; DMEM, Dulbecco's modified Eagle's medium; TK, thymidine kinase; at Columbia University, were grown in DMEM supplemented with 2 PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; hGH, human growth hormone; X-gal, 5-bromo-7-chloro-3-indolyl-~-i)-galactopyrano- mM glutamine and 10% bovine calf serum. To derive stable transfor- side; ONPG, O-nitrophenyl-/3-o-galactopyranoside; FITC, fluorescein isothiocyo- mants, cells were transfected with 10:1 mixtures of the reporter gene nate; IgG, immunoglobulin G; CHP, 4-cis-hydroxy-L-proline. plasmid (see below and Fig. 1) and plasmid pIBW DNA containing a 6877 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1992 American Association for Cancer Research. REGULATION OF SMOOTH MUSCLE a-ACTIN PROMOTER IN ras-TRANSFORMED CELLS neomycin resistance marker fused to the herpes simplex virus TK pro- were washed extensively with PBS solution and photographed using a moter. Briefly, 5 x 105 cells, plated in 100-mm dishes, were incubated Zeiss IM-35 fluorescent microscope equipped with a 200-W mercury for 6 h with calcium phosphate DNA precipitates. Calcium phosphate light source. precipitates were prepared with 20 ~tg of plasmid DNA per 10-cm plate (15). The transfection was stopped by replacing the medium with the Isolation and Characterization of Revertant Cell Line standard culture medium after a brief rinse with PBS. Twenty-four h later, transfected cultures were split 1:10 and grown in the presence of To isolate the revertant cell line D-3, Rat-6 (2S-HO6) cells were the neomycin analogue G418 (Sigma) at 400 #g/ml. After 10 days, seeded at a density of 1 x 106 cells/100-mm dish and treated with 7 resistant clones were isolated using cloning cylinders. ~g/ml of 5-aza-cytidine for 24 h. After a recovery period of an addi- tional 48 h, cells were plated at a density of 1 x 105 cells for each 100-mm dish in the growth medium containing 200/~g/ml of CHP Construction of Plasmids (23, 24). The plates were fluid changed with DMEM containing CHP, The EcoRI-DraIII fragment consisting of-894 to + 12 base pairs of twice weekly for 3 to 4 wk, after which colonies made up of apparently the human a-actin gene was isolated from the paAS plasmid (16) and nontransformed cells were marked and isolated using cloning cylinders. ligated into the HindIII site of plasmids pCH 126 (17), pOGH (18), and Cells were further subcloned using the limiting dilution technique. pBasic (19) to derive paAP126, paAPGH, and paAPCAT plasmids, To determine the susceptibility of cells for transformation by retro- respectively. The structure of the plasmids was confirmed by DNA viruses containing Kirsten and Harvey ras oncogenes, Rat-6 (2S) and sequence analysis, pBasic and pOGH are promoterless plasmids con- D-3 revertant cells were plated at a density of 2 x 105 ceils in 100-mm taining CAT and hGH coding sequences, respectively (18, 19). Plasmid dishes, and virus stocks (a gift from Dr. Bassin, National Cancer Insti- pHO6T1 containing the T24 Ha-ras oncogene was a kind gift from Dr. tute) diluted with the growth medium were added to the cells in the Earl Ruley (Massachusetts Institute of Technology) (20). presence of 200 ug/ml of DEAE-dextran. Following infection for 2 h, the medium containing the virus was aspirated and replaced with fresh medium. The cells were fed with fresh medium at 3-day intervals, and Enzyme Assays transformed foci were scored after 12 days by fixing the cells in meth- /~-Galactosidase Assay. To identify stable fibroblast cell lines ex- anol and staining with Giemsa reagent as described (24).
Load more

Recommended publications
  • Biochemistrystanford00kornrich.Pdf
    University of California Berkeley Regional Oral History Office University of California The Bancroft Library Berkeley, California Program in the History of the Biosciences and Biotechnology Arthur Kornberg, M.D. BIOCHEMISTRY AT STANFORD, BIOTECHNOLOGY AT DNAX With an Introduction by Joshua Lederberg Interviews Conducted by Sally Smith Hughes, Ph.D. in 1997 Copyright 1998 by The Regents of the University of California Since 1954 the Regional Oral History Office has been interviewing leading participants in or well-placed witnesses to major events in the development of Northern California, the West, and the Nation. Oral history is a method of collecting historical information through tape-recorded interviews between a narrator with firsthand knowledge of historically significant events and a well- informed interviewer, with the goal of preserving substantive additions to the historical record. The tape recording is transcribed, lightly edited for continuity and clarity, and reviewed by the interviewee. The corrected manuscript is indexed, bound with photographs and illustrative materials, and placed in The Bancroft Library at the University of California, Berkeley, and in other research collections for scholarly use. Because it is primary material, oral history is not intended to present the final, verified, or complete narrative of events. It is a spoken account, offered by the interviewee in response to questioning, and as such it is reflective, partisan, deeply involved, and irreplaceable. ************************************ All uses of this manuscript are covered by a legal agreement between The Regents of the University of California and Arthur Kornberg, M.D., dated June 18, 1997. The manuscript is thereby made available for research purposes. All literary rights in the manuscript, including the right to publish, are reserved to The Bancroft Library of the University of California, Berkeley.
    [Show full text]
  • Suppression of Oncogenic Ras by Mutant Neurofibromatosis Type 1 Genes with Single Amino Acid Substitutions
    Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6706-6710, July 1993 Biochemistry Suppression of oncogenic Ras by mutant neurofibromatosis type 1 genes with single amino acid substitutions MASATO NAKAFUKU*t, MASARU NAGAMINE*, AKIHIRA OHTOSHI*, KAZUMA TANAKAO§, AKIo TOH-E*, AND YOSHITO KAZIRO*¶ *DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304-1104; and tDepartment of Biology, Faculty of Science, University of Tokyo, Tokyo 113, Japan Communicated by Paul Berg, April 1, 1993 ABSTRACT NFI was frit identified as the gene responsible growth through the regulation of adenylate cyclase (14, 15). for the pathogenesis of the human genetic disorder neurofibro- Thus, yeast cells carrying activated mutations in Ras (such as matosis type 1. cDNA cloning revealed that its putative protein [Val'9]Ras2 and [Leua]Ras2) are defective in responding to product has a domain showing signfifcant sequence homology environmental conditions and show a variety of phenotypes with the mammalian Ras GTPase activating protein and two including a heat-shock-sensitive phenotype (14, 15). yeast Saccharomyces cerevisiae proteins, Iral and Ira2. The Ras S. cerevisiae also possesses two NF1 homologs, Iral and GTPase activating protein-related domain of the NFI gene Ira2, and human NF1 is structurally closer to yeast Ira than product (NF1-GRD) stimulates GTPase activity of normal Ras to human GAP (1-4). NF1 and Ira are also functionally proteins but not of oncogenic mutant Ras from both mamma- related since NF1-GRD expressed in yeast cells can com- lian and yeast cells. Thus, in yeast, NF1-GRD can suppress the plement ira-deficient yeast.
    [Show full text]
  • Colony-Stimulating Factor
    Proc. Natl. Acad. Sci. USA Vol. 83, pp. 7633-7637, October 1986 Biochemistry Isolation and characterization of the cDNA for murine granulocyte colony-stimulating factor (murine fibrosarcoma NFSA cells/cDNA sequence/protein homology/growth factor/differentiation) MASAYUKI TSUCHIYA, SHIGETAKA ASANO, YOSHITo KAZIRO, AND SHIGEKAZU NAGATA Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan Communicated by Charles Yanofsky, June 26, 1986 ABSTRACT A cDNA sequence coding for murine granu- In this report, we describe the isolation of the murine locyte colony-stimulating factor (G-CSF) has been isolated G-CSF cDNA from a recombinant X phage library prepared from a cDNA library prepared with mRNA derived from from the murine fibrosarcoma NFSA cell line, which pro- murine fibrosarcoma NFSA cells, which produce G-CSF con- duces G-CSF constitutively. The murine G-CSF cDNA was stitutively. Identification of murine G-CSF cDNA was based on identified by using cross-hybridization with human G-CSF the cross-hybridization with human G-CSF cDNA under a cDNA under a low-stringency condition. The cDNA was low-stringency condition. The cDNA can encode a polypeptide expressed in monkey COS cells under the simian virus 40 consisting of a 30-amino acid signal sequence, followed by a (SV40) early promoter. The protein produced by COS cells mature G-CSF sequence of 178 amino acids with a calculated had an ability to stimulate the granulocyte colony formation Mr of 19,061. The nucleotide sequence and the deduced amino in bone marrow cells and to support the proliferation of acid sequence of murine G-CSF cDNA were 69.3% and 72.6% murine NFS-60 myeloid leukemic cells.
    [Show full text]
  • Thrombin-Induced Inhibition of Myoblast Differentiation Is Mediated
    FEBS 23593 FEBS Letters 472 (2000) 297^301 CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Thrombin-inducedConnector inhibition of myoblast di¡erentiation is mediated by GLQ Motoshi Nagao, Yoshito Kaziro, Hiroshi Itoh* Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan Received 17 March 2000 Edited by Julio Celis an important role in skeletal muscle development. It has been Abstract Thrombin has been shown to inhibit skeletal muscle differentiation. However, the mechanisms by which thrombin shown that thrombin is involved in synapse elimination occur- represses myogenesis remain unknown. Since the thrombin ring at the neuromuscular junction during muscle develop- ment [9,10]. In addition, thrombin causes a delay in skeletal receptor couples to Gi,Gq=11 and G12, we examined which subunits of heterotrimeric guanine nucleotide-binding regulatory myogenesis [4^7]. The thrombin receptor is expressed in cul- proteins (GKi,GKq=11,GK12 or GLQLQ) participate in the thrombin- tured myoblasts and its expression decreases once the myo- induced inhibition of C2C12 myoblast differentiation. GKi2 and blasts fuse to form multinucleated myotubes [5]. Thrombin GK11 had no inhibitory effect on the myogenic differentiation. has been reported to act as a mitogen in myoblasts as well GK12 prevented only myoblast fusion, whereas GLQLQ inhibited both as ¢broblasts and smooth muscle cells, and induce their pro- the induction of skeletal muscle-specific markers and the liferation [4,5]. In contrast, it has been reported that thrombin myotube formation. In addition, the thrombin-induced reduction does not induce a mitogenic signal in myoblastic cell line [6], of creatine kinase activity was blocked by the C-terminal peptide of L-adrenergic receptor kinase, which is known to sequester free and that it inhibits myoblast apoptosis and functions as a GLQLQ.
    [Show full text]
  • Saccharomyces Cerevisiae 1
    Characterization of the Alpha- and Beta-Tubulin Polypeptides in Saccharomycescerevisiae by Vida Praitis B.A., Biology. Swarthmore College, 1988 Submitted to the Department of Biology in Partial Fulfillment of the Requirements for the Degree of Doctor of Science in Biology at the Massachusetts Institute of Technology June 1995 ©1995 Vida Praitis. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part. Signature of Author ........................................... Department of Biology Certified by................... ................................................................................................. Professor Frank Solomon Thesis Supervisor Accepted by ........................................................................ ......... .............. Professor Frank Solomon Chairman, Graduate Thesis Committee Science MASSACHIJSETTSINSTITUTE OF TF mF.lil tyV 'JUN 06 1995 ICIDAli-.u Characterization of the Alpha- and Beta-Tubulin Polypeptides in Saccharomycescerevisiae by Vida Praitis Submitted to the Department of Biology on May 5, 1995 in partial fulfillment of the requirements for the Degree of Doctor of Science in Biology Abstract: Microtubules, involved in a number of critical, diverse cellular structures and functions, are composed primarily of two related but non-identical, highly conserved subunits, alpha- and beta-tubulin. The precise secondary and tertiary interactions of the tubulin subunits are still poorly understood, likely because native alpha- or beta-tubulin has not been purified in large enough quantities to perform structural or biochemical analysis. Crystal structures, which would provide extensive structural information about these molecules and their interactions, have not been reported. I sought to characterize the alpha- and beta-tubulin polypeptides using two approaches, one genetic and one biochemical. E. Raff's laboratory characterized a series of mutations in the testes-specific beta-tubulin of D.
    [Show full text]
  • Toward a Comprehensive Model of a Yeast Stress Response Pathway
    TOWARD A COMPREHENSIVE MODEL OF A YEAST STRESS RESPONSE PATHWAY Patrick Charles McCarter A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum in Bioinformatics and Computational Biology. Chapel Hill 2017 Approved by: Timothy Elston Beverly Errede Henrik Dohlman Shawn Gomez Barry Lentz Laura Miller ©2017 Patrick Charles McCarter ALL RIGHTS RESERVED ii ABSTRACT Patrick Charles McCarter: Toward a comprehensive model of a yeast stress response pathway (Under the direction of Timothy Elston) Cells rely on cyto-protection programs to survive exposure to external stressors. In eukaryotes, many of these programs are regulated by Mitogen-Activated Protein Kinases (MAPK). Aberrant signaling in MAPK pathways is associated with pathologies including cancer and neurodegenerative disease. Thus understanding how MAPKs are dynamically regulated is critical for human health. We leverage the simplicity and genetic tractability of S. cerevisiae (yeast) to experimentally investigate and mathematically model MAPK activity in the prototypical High Osmolarity Glycerol (HOG) pathway. The HOG pathway transmits osmostress to Hog1, a MAPK which is homologous to the p38 and JNK kinases, via two distinct signaling branches (Sho1, Sln1). Once phosphorylated, Hog1 induces activation of the hyper-osmotic stress adaptation program. Our goal was to identify and characterize the feedback network that regulates Hog1 activity during hyper-osmotic stress. We previously demonstrated that Hog1 phosphorylation is encoded via positive feedback, and in agreement with previous studies, we showed that Hog1 dephosphorylation is encoded via negative feedback. We used mathematical modeling to define simplified MAPK feedback networks that featured various combinations of negative and positive feedback loops.
    [Show full text]
  • 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
    Proc. Natl. Acad. Sci. USA Vol. 88, pp. 3314-3318, April 1991 Biochemistry 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 (growth factor receptor/lymphokine/cytokine) TAKAYA SATOH, MASATO NAKAFUKU, ATSUSHI MIYAJIMA, AND YOSHITO KAZIRO DNAX Research Institute of Molecular and Cellular Biology, 901 California Avenue, Palo Alto, CA 94304-1104 Communicated by Joseph L. Goldstein, January 7, 1991 ABSTRACT The protooncogene ras acts as a component of activation of ras p21 (7). In this case, the involvement of signal-transduction networks in many kinds of cells. The ras tyrosine kinases in signal transduction is not known. gene product (p21) is a GTP-binding protein, and the activity A variety of cytokines are involved in survival, prolifera- ofthe protein is regulated by bound GDP/GTP. Recent studies tion, and differentiation of hematopoietic cells. Recently, have shown that a certain class ofgrowth factors stimulates the cDNAs ofvarious cytokine receptors were cloned, and it has formation of active p2lFGTP complexes in fibroblasts and that been revealed that cytokine receptors including those for oncogene products with enhanced tyrosine kinase activities interleukin 2 (IL-2), IL-3, IL-4, and granulocyte/macrophage have a similar effect on ras p21. We have measured the ratio colony-stimulating factor (GM-CSF) are members of a re- of active GTP-bound p21 to total p21 in several lymphoid and ceptor family that are distinct from growth factor receptors myeloid cell lines in order to understand the role of ras in the with a tyrosine kinase and from hormonal receptors coupled proliferation of these cells.
    [Show full text]
  • EMOTO, Kazunori1, WADA, Takeo1, KUTSUKAKE, Kazuhiro1 (1Grad
    Genes Genet. Syst. (2009) 84 477 Symposia (S1-1 – S2-10) 3E EMOTO, Kazunori1, WADA, Takeo1, KUTSUKAKE, S1 IKEDA, Masato1 (1Dept. BioSci. and BioTech., Faclt. -11 Kazuhiro1 (1Grad. Sch. Natural Sci. Tech., Okayama -1 Agr., Shinshu Univ.) Univ.) Genome-based strain reconstruction for amino acid produc- Target protein of the RpoS-induced toxin in Salmonella tion enterica serovar Typhimurium The classical approach to strain improvement involves random Although toxin-antitoxin (TA) systems are ubiquitously present mutagenesis and screening, but this technology cannot avoid on bacterial chromosomes, their biological significance has accumulating detrimental or unnecessary mutations. Genome- remained enigmatic. We previously reported that Salmonella based strain reconstruction is a technology that identifies the enterica serovar Typhimurium possesses a toxin gene rsaB, useful mutations in such strains through genome sequencing and which is induced by a stress-specific sigma factor RpoS and then systematically and precisely engineers those mutations into causes growth arrest. Together with its cognate antitoxin gene the wild-type genome, creating a strain with only the useful rsaA, this gene seems to constitute a TA system on the mutations. Strains derived by this reverse engineering method Salmonella chromosome. In this study, as the first step to are expected to be more robust, give higher fermentation yields in elucidate the physiological role of this TA system, we attempted a shorter time and resist stressful conditions. Here we describe to identify a target protein of the toxin. From the cell lysate of the technology by using L-lysine fermentation as a model. Salmonella, an RNA-binding protein CsrA was found to be co- purified with the GST-tagged RsaB protein.
    [Show full text]
  • Association of the Proto-Oncogene Product Dbl with G Protein LQ Subunits
    FEBS Letters 459 (1999) 186^190 FEBS 22684 Association of the proto-oncogene product Dbl with G protein LQ subunits Kazuhiko Nishida, Yoshito Kaziro, Takaya Satoh* Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan Received 13 August 1999; received in revised form 3 September 1999 dem, designated Dbl homology (DH) and pleckstrin homol- Abstract The Rho family of GTP-binding proteins has been implicated in the regulation of various cellular functions ogy (PH) domains. The DH domain is essential and su¤cient including actin cytoskeleton-dependent morphological change. for GEF activity, whereas the PH domain determines proper Its activity is directed by intracellular signals mediated by cellular localization thereby modulating the function of the various types of receptors such as G protein-coupled receptors. DH domain. Some Dbl family members are speci¢c for an However, the mechanisms underlying receptor-dependent regula- individual Rho family protein, while others act on all three tion of Rho family members remain incompletely understood. subgroups. Dbl is a prototype of this family, which acts on The guanine nucleotide exchange factor (GEF) Dbl targets Rho Cdc42, Rac1, and RhoA in vitro, and is expressed in brain, family proteins thereby stimulating their GDP/GTP exchange, adrenal gland, testis, and ovary [4^6]. Truncation of the ami- and thus is believed to be involved in receptor-mediated no-terminal half enables Dbl to transform NIH3T3 cells, sug- regulation of the proteins. Here, we show the association of gesting that the amino-terminal region is involved in the reg- Dbl with G protein LQLQ subunits (GLQLQ) in transient co-expression and cell-free systems.
    [Show full text]
  • Ha-Ras and N-Ras Regulate MAPK Activity by Distinct Mechanisms in Vivo
    Oncogene (1998) 16, 1417 ± 1428 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 Ha-ras and N-ras regulate MAPK activity by distinct mechanisms in vivo Mark Hamilton and Alan Wolfman Department of Cell Biology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA The Ras GTPases function as molecular switches, sucient for Raf-1 activation (Stokoe and McCormick, regulating a multiplicity of biological events. However 1997). Raf activity mediates the activation of the the contribution, if any, of a speci®c c-Ras isoform (Ha-, MAPK cascade through the Raf-dependent activation N-, or Ki-ras A or B) in the regulation of a given of MEK1 (Macdonald et al., 1993; Moodie et al., 1993, biological or biochemical process, is unknown. Murine 1994; Van Aelst et al., 1993), the regulatory kinase for C3H10T1/2 ®broblasts transformed with activated the p42 and p44 MAPKs. MAPK regulates the activity (G12V)Ha-ras or (Q61K)N-ras proliferate in serum-free of cytosolic kinases, including PLA2 (Lin et al., 1993, media and have constitutive MAPK activity. The growth and RSK2 (Xing et al., 1996) and, upon translocation factor antagonist, suramin, inhibited the serum-indepen- of active MAPK to the nucleus, the activity of dent proliferation of Ha-ras transformed ®broblasts, but transcription factors including Elk1 (reviewed by Hill not the serum-independent proliferation of N-ras trans- and Treisman, 1995). Ras activity is critical for formed cells. The inhibition of cell proliferation was proliferation since both the microinjection of neutraliz- concomitant with the abrogation of the constitutive ing Ras antibodies (Mulcahy et al., 1985) or the ectopic MAPK activity in the Ha-ras transformed ®broblasts.
    [Show full text]
  • 2014 World Alliance Forum in San Francisco the Impact Of
    Organization in Special Consultative Status with the UN Economic and Social Council www.allianceforum.org/en 2014 World Alliance Forum in San Francisco The Impact of Regenerative Medicine 2014 Mission Bay Conference Center at UCSF November 6 - 7, 2014 Organized by: Alliance Forum Foundation Government of Japan (Consulate General of Japan in San Francisco) www.wafsf.org Table of Contents Page Greetings 3 Program 6 Speakers’ Biography 12 Masters of Ceremonies’ Biography 26 Sponsors & Major Supporting Organization 27 2 Greetings Organization in Special Consultative Status with the UN Economic and Social Council Greetings from George Hara Chairman of the Board, Alliance Forum Foundation The Alliance Forum Foundation began holding its annual World Alliance Forum in the 1990s with a goal to nurture emerging technologies in the fields of IT and healthcare. Since then, the Forum has continued to be a catalyst for many new alliances among businesses, research institutions, governments, and local and international organizations that enable commercialization of innovative technologies. Last year, at the 2013 Forum, the Foundation invited leading researchers engaged in stem cell research in Japan and the US to come together in San Francisco. In the festive atmosphere of celebrating Dr. Shinya Yamanaka and his 2012 Nobel Prize in Physiology or Medicine, participants gave presentations on this new stage in the development of regenerative medicine made possible by Dr. Yamanaka's discovery of induced pluripotent stem cells (iPSC). It became clear at
    [Show full text]
  • Saccharomyces Cerevisiae
    Proc. Nati. Acad. Sci. USA Vol. 80, pp. 6192-6196, October 1983 Biochemistry Molecular cloning and sequence determination of the nuclear gene coding for mitochondrial elongation factor Tu of Saccharomyces cerevisiae (protein synthesis/tuf gene probe/Southern hybridization/sequence conservation) SHIGEKAZU NAGATA, YASUKO TSUNETSUGU-YOKOTA, AYAKO NAITO, AND YOSHITo KAZIRO Institute of Medical Science, University of Tokyo, Minatoku, Takanawa, Tokyo 108, Japan Communicated by Severo Ochoa, July 8, 1983 ABSTRACT A 3. 1-kilobase Bgl H fragment of Saccharomyces functions in the mitochondrial translational apparatus (16, 17). cerevisiae carrying the nuclear gene encoding the mitochondrial Translational factors as well as ribosomal proteins in the mi- polypeptide chain elongation factor (EF) Tu has been cloned on tochondria are encoded by nuclear genes, synthesized in cy- pBR327 to yield a chimeric plasmid pYYB. The identification of toplasmic fractions, and transported into the mitochondria (18). the gene designated as tufM was based on the cross-hybridization The translational machineries of mitochondria have been thought with the Escherichia coli tufB gene, under low stringency con- to be closer to the prokaryotic ones than to the machinery pres- ditions. The complete nucleotide sequence of the yeast tufM gene ent in eukaryotic cytoplasm (17, 19). However, the organization was established together with its 5'- and 3'-flankdng regions. The of the nuclear for the mitochondrial translational sequence contained 1,311 nucleotides coding for a protein of 437 genes appa- amino acids with a calculated Mr of 47,980. The nucleotide se- ratus, their expression, and the incorporation of the products quence and the deduced amino acid sequence of tufM were 60% into the mitochondria are not well understood.
    [Show full text]