CSM News Electronic Edition Volume 4, number 7 February 25, 1995 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmsbio.nwu.edu/dicty.html" =========== Abstracts =========== Identification of a UDPGlcNAc: Serine N-acetylglucosamine-1-phosphate transferase in Dictyostelium discoideum. An enzyme that initiates phosphoglycosylation. Hudson H. Freeze and Mie Ichikawa La Jolla Cancer Research Foundation Biochemical and Biophysical Research Communications, in press ABSTRACT A lysosomal proteinase from Dictyostelium discoideum was previously shown to have GlcNAc alpha-1-P residues in phosphodiester linkage to serine. We have identified a GlcNAc-1-P transferase activity in membrane preparations using UDP[3H]GlcNAc and a peptide acceptor with two Ser-Gly tandem repeats. We established an assay, proved the structure of the product, determined the Kms for donor and acceptor and showed that the glycopeptide binds a GlcNAc-1-P specific rabbit antibody. These findings provide the basis to search for mutants lacking GlcNAc-1-P transferase activity as a probe for the function of this modification which we call phosphoglycosylation. ----------------------------------------------------------------------- A density-sensing factor regulates signal transduction in Dictyostelium Ita S. Yuen1, Renu Jain1, John D. Bishop1, David F. Lindsey1,3, William J. Deery1, Peter J. M. Van Haastert2, and Richard H. Gomer1,3 1Department of Biochemistry and Cell Biology and 3Howard Hughes Medical Institute, Rice University, Houston, TX 77251-1892 USA, and 2Biochemistry Department,University of Groningen, Nijenborgh 49747 AG Groningen The Netherlands J. Cell Biology, in press Abstract Dictyostelium discoideum initiates development when cells overgrow their bacterial food source and starve. To coordinate development, the cells monitor the extracellular level of a protein, CMF, secreted by starved cells. When a majority of the cells in a given area have starved, as signalled by CMF secretion, the extracellular level of CMF rises above a threshold value and permits aggregation of the starved cells. The cells aggregate using relayed pulses of cAMP as the chemoattractant. Cells in which CMF accumulation has been blocked by antisense do not aggregate except in the presence of exogenous CMF. We find that these cells are viable but do not chemotax towards cAMP. Videomicroscopy indicates that the inability of CMF antisense cells to chemotax is not due to a gross defect in motility, although both video and scanning electron microscopy indicate that CMF increases the frequency of pseudopod formation. The activations of Ca++ influx, adenylyl cyclase and guanylyl cyclase in response to a pulse of cAMP are strongly inhibited in cells lacking CMF, but are rescued by as little as 10 seconds exposure of cells to CMF. The activation of phospholipase C by cAMP is not affected by CMF. Northern blots indicate normal levels of the cAMP receptor mRNA in CMF antisense cells during development, while cAMP binding assays and Scatchard plots indicate that CMF antisense cells contain normal levels of the cAMP receptor. In Dictyostelium, both adenylyl and guanylyl cyclases are activated via G proteins. We find that the interaction of the cAMP receptor with G proteins in vitro is not measurably affected by CMF, whereas the activation of adenylyl cyclase by G proteins requires cells to have been exposed to CMF. CMF thus appears to regulate aggregation by regulating an early step of cAMP signal transduction. -------------------------------------------------------------------- STIMULATION OF CALCIUM INFLUX BY PLATELET ACTIVATING FACTOR IN DICTYOSTELIUM Ralph Schaloske1, Concetta Sordano2, Salvatore Bozzaro2* and Dieter Malchow1 1Faculty of Biology, University of Konstanz, Postfach 5560, 78462 Konstanz (FRG) and 2Department of Clinical and Biological Sciences, University of Turin, Ospedale San Luigi, 10043 Orbassano-Torino (Italy) J. Cell Science, in press. Abstract Platelet activating factor (PAF) induces Ca2+ influx in Dictyostelium discoideum. In this investigation we used this activity to analyze the mechanism of PAF action. We found that PAF activity was confined to the period of spike-shaped oscillations and suggest that the role of PAF is to augment cAMP relay. PAF seems to act only a few times during this time period of two hours, since Ca2+ entry adapted to a subsequent stimulus for about 30 min. PAF showed a reduced response in the G-protein b- strain LW14 and was unable to induce Ca2+ influx in the Ga2- strains HC85 and JM1. The latter expresses the cAMP receptors cAR1 constitutively, and exhibits cAMP-induced Ca2+ influx albeit at a reduced level. In order to decide whether the inability of PAF to elicit a Ca2+ response in JM1 cells was due to the lack of differentiation and/or the lack of Ga2 we inhibited the IP3-dependent pathway with compound U73122 and found that Ca2+ entry was blocked, whereas a closely related inactive compound, U73343, did not alter the response. In agreement with this NBD-Cl, an inhibitor of Ca2+ uptake into the IP3-sensitive store in Dictyostelium, also abolished PAF activity. The latter was not inhibited by the plasma membrane antagonists BN-52021 or WEB 2170. Therefore PAF seems to operate intracellularly via the IP3-signalling pathway at or upstream of the IP3-sensitive store. ---------------------------------------------------------------------- [End CSM-News, volume 4, number 7]