Dicty News Electronic Edition Volume 12, number 12 June 12, 1999 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" ============= Abstracts ============= Evidence for the presence of a NF-kB signal transduction system in Dictyostelium discoideum Francois TRAINCARD1, Eleonora PONTE1, Jason PUN2, Barrie COUKELL2 and Michel VERON1 1: Unité de Régulation enzymatique des Activités cellulaires, CNRS URA 1773, Institut Pasteur, 25 rue du Dr. Roux, 75724, Paris Cedex 15, France. 2: York University, 4700 Keele St., Toronto, Ontario, M3J 1P3, Canada. SUMMARY The Rel/NF-kB family of transcription factors and regulators has been described so far only in vertebrates and arthropods where they mediate responses to many extracellular signals. No counterparts of genes coding for such proteins have been identified in the Caenorhabditis elegans genome and no NF-kB activity was found in Saccharomyces cerevisiae. We describe here the presence of an NF-kB transduction pathway in the lower eukaryote Dictyostelium discoideum. Using antibodies raised against components of the mammalian NF-kB pathway, we demonstrate the presence in Dictyostelium extracts of proteins homologous to Rel/NF-kB, IkB and IKK components. Using gel shift experiments, we demonstrate the presence in nuclear extracts of developing Dictyostelium cells of proteins binding to kB consensus oligonucleotides and to a GC-rich kB-like sequence, lying in the promoter of cbpA, a developmentally regulated Dictyostelium gene encoding the Ca2+-binding protein CBP1. Using immunofluorescence, we show a specific nuclear translocation of the p65 and p50 homologues of the NF-kB transcription factors as vegetatively growing cells develop to the slug stage. Taken together, our results strongly indicate the presence of a complete NF-kB signal transduction system in Dictyostelium discoideum that could be involved in the developmental process. ---------------------------------------------------------------------------- Intracellular ribonuclease isozyme activity of the cellular slime mold cells Dictyostelium discoideum Ax2 and Dictyostelium mucoroides Saburo Uchiyama, Shin-Ichi Nagai, Norio Inokuchi,1 Takashi Koyama1 and Keizo Maruyama2 Laboratory of Biology, Dokkyo University School of Medicine, Mibu, Tochigi 3 21-0293, 1Department of Microbiology, College of Pharmacy, Nihon University, Narashinodai 7-7-1, Funabashi, Chiba 274-0063, and 2Laboratory of Biology, F aculty of Integrated Human Studies, Kyoto University, Kyoto 606-8501, Japan J. Basic Microbiol. (in press) Summary The isozymes of ribonuclease were analyzed in cell-free, crude extracts of cells by reversed activity staining of polyacrylamide gradient gels after electrophoresis. We previously reported three isozymes of the intracellular ribonuclease in Dictyostelium discoideum NC4, which were found in vegetative cells. In the present study, we examined the ribonucleolytic activity of an axenic strain of D. discoideum Ax2 cells which can grow with or with out bacteria. The mobility of these isozymes (Dd I, II and III) was the same regardless of the difference in the feeding condition. We also found the three isozymes of D. mucoroides cells, Dm I, II and III. Dm I was different from Dd I in D. discoideum cells, while Dm II and Dm III were the same as Dd II and Dd III in terms of the mobility in polyacrylamide gradient gels during electrophoresis. ---------------------------------------------------------------------------- Loss of a prolyl oligopeptidase confers resistance to lithium by elevation of Inositol (1,4,5) trisphosphate. R.S.B. Williams, M. Eames, J. Ryves, J. Viggars & A.J. Harwood* MRC Laboratory for Molecular Cell Biology, University College London Gower St., London, WC1E 6BT EMBO J., in press. Abstract The therapeutic properties of lithium ions (Li+) are well known, however the mechanism of its action remains unclear. To investigate this problem, we have isolated Li+ resistant mutants from Dictyostelium. In this paper, we describe the analysis of one of these mutants. This mutant lacks the Dictyostelium prolyl oligopeptidase gene (dpoA). We have examined the relationship between dpoA and two major biological targets of lithium: glycogen synthase kinase 3 (GSK-3) and signal transduction via inositol (1,4,5) trisphosphate (IP3). We find no evidence for an interaction with GSK-3, but instead find that loss of dpoA causes an increased concentration of IP3. The same increase in IP3 is induced in wild type cells by a prolyl oligopeptidase inhibitor. IP3 concentrations rise via an unconventional mechanism that involves enhanced dephosphorylation of inositol (1,3,4,5,6) pentakisphosphate (IP5). Loss of DpoA activity therefore counteracts the reduction in IP3 concentration caused by Li+ treatment. Abnormal prolyl oligopeptidase (POase) activity is associated with both unipolar and bipolar depression, however the function of POase in these conditions is unclear. Our results offer a novel mechanism that links prolyl oligopeptidase activity to IP3 signalling and provides further clues for the action of Li+ in the treatment of depression. ---------------------------------------------------------------------------- ROLE OF THE SALT-BRIDGE BETWEEN SWITCH-1 AND SWITCH-2 OF DICTYOSTELIUM MYOSIN M. Furch, S. Fujita-Becker, M.A. Geeves*, K.C. Holmes, & D.J. Manstein Max-Planck-Institute for Medical Research, Dept. of Biophysics, Jahnstrasse 29, D-69120 Heidelberg and *Max-Planck-Institut für Molekulare Physiologie, Postfach 102664, D-44026 Dortmund, Germany J. Mol. Biol., in press. ABSTRACT Motifs N2 and N3, also referred to as switch-1 and switch-2, form part of the active site of molecular motors such as myosins and kinesins. In the case of myosin, N3 is thought to act as a gamma-phosphate sensor and moves almost 0.6 nm relative to N2 during the catalysed turnover of ATP, opening and closing the active site surrounding the gamma-phosphate. The closed form seems to be necessary for hydrolysis and is stabilised by the formation of a salt-bridge between an arginine in N2 and a glutamate in N3. We examined the role of this salt-bridge in Dictyostelium discoideum myosin. Myosin motor domains with mutations E459R or R238E, that block salt-bridge formation, show defects in nucleotide-binding, reduced rates of ATP hydrolysis and a 10-fold reduction in actin affinity. Inversion of the salt-bridge in double-mutant M765-IS eliminates most of the defects observed for the single- mutants. With the exception of a 2,500-fold higher Km for ATP, the double- mutant displayed enzymatic and functional properties very similar to those of the wild-type protein. Our results reveal that, independent of its orientation, the salt-bridge is required to support efficient ATP hydrolysis, normal communication between different functional regions of the myosin head, and motor function. ---------------------------------------------------------------------------- FUNCTIONAL CHARACTERISATION OF DICTYOSTELIUM MYOSIN II WITH CONSERVED TRYPTOPHANYL RESIDUE 501 MUTATED TO TYROSINE Renu Batra and Dietmar J. Manstein Max-Planck-Institut für Medizinische Forschung, Jahnstr. 29, D-69120 Heidelberg, Germany Biol. Chem., in press. ABSTRACT We created a Dictyostelium myosin II mutant in which the highly conserved residue Trp-501 was replaced by a tyrosine residue. The mutant myosin alone, when expressed in a Dictyostelium strain lacking the functional myosin II heavy chain gene, supported cytokinesis and multicellular development, processes which require a functional myosin in Dictyostelium. Additionally, we expressed the W501Y mutant in the soluble myosin head fragment M761-2R (W501Y-2R) to characterise the kinetic properties of the mutant myosin motor domain. The affinity of the mutant myosin for actin was approximately 6-fold decreased but other kinetic properties of the protein were changed less than 2-fold by the W501Y mutation. Based on spectroscopic studies and structural considerations Trp-501, corresponding to Trp-510 in chicken fast skeletal muscle myosin, has been proposed to be the primary ATP-sensitive tryptophanyl residues. Our results confirm these conclusions. While the wild-type construct displayed a 10% fluorescence increase, addition of ATP to W501Y-2R was not followed by an increase in tryptophan fluorescence emission. ---------------------------------------------------------------------------- A Serpentine Receptor-dependent, Gbeta- and Ca2+ Influx-independent Pathway Regulates Mitogen-activated Protein Kinase ERK2 in Dictyostelium Paul W. Schenk, Thomas Nebl, Paul R. Fisher and B. Ewa Snaar-Jagalska Section of Cell Biology, Institute of Molecular Plant Sciences, Leiden University, P.O. Box 9505, 2300 RA Leiden, The Netherlands and School of Microbiology, La Trobe University, Bundoora, VIC 3083, Australia Biochem. Biophys. Res. Commun., in press Abstract Ca2+ influx and mitogen-activated protein (MAP) kinase activation are important phenomena in signal transduction, which are often interconnected. We investigated whether serpentine receptor-dependent, G beta-independent activation of MAP kinase ERK2 by chemoattractant cyclic AMP (cAMP) is mediated by Ca2+ influx in the social amoeba Dictyostelium discoideum. We generated a D. discoideum double mutant, which harbours a temperature- sensitive G beta subunit and expresses the apoaequorin protein. Utilizing this mutant, we demonstrate that cAMP induced Ca2+ influx into intact D. discoideum cells can be blocked completely at both the permissive and the restrictive temperature, by using either gadolinium ions or Ruthenium Red. Under the same experimental conditions, these substances do not abolish cAMP stimulation of ERK2 at either temperature. We conclude that there is a G beta- and Ca2+ influx-independent pathway for the receptor-dependent activation of MAP kinase ERK2 in Dictyostelium discoideum. ---------------------------------------------------------------------------- Signal perception and transduction: the role of protein kinases Paul W. Schenk and B. Ewa Snaar-Jagalska Section of Cell Biology, Institute of Molecular Plant Sciences, Leiden University, P.O. Box 9505, 2300 RA Leiden, The Netherlands Biochim. Biophys. Acta 1449 (1999) 1-24 Abstract Cells can react to environmental changes by transduction of extracellular signals, to produce intracellular responses. Membrane-impermeable signal molecules are recognized by receptors, which are localized on the plasma membrane of the cell. Binding of a ligand can result in the stimulation of an intrinsic enzymatic activity of its receptor or the modulation of a transducing protein. The modulation of one or more intracellular transducing proteins can finally lead to the activation or inhibition of a so-called ‘effector protein'. In many instances, this also results in altered gene expression. Phosphorylation by protein kinases is one of the most common and important regulatory mechanisms in signal transmission. This review discusses the non-channel transmembrane receptors and their downstream signaling, with special focus on the role of protein kinases. ---------------------------------------------------------------------------- [End Dicty News, volume 12, number 12]