CSM News Electronic Edition Volume 8, number 11 May 3, 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =========== Abstracts =========== SH2 signalling in a lower eukaryote: a STAT protein that regulates stalk cell differentiation in Dictyostelium Takefumi Kawata*, Andrej Shevchenko+, Masashi Fukuzawa*, Keith A. Jermyn*, Nicholas F. Totty§, Natasha V. Zhukovskaya*, Alistair E. Sterling§, Matthias Mann+ and Jeffrey G. Williams* MRC Laboratory of Molecular Cell Biology, and Department of Biology, University College London, Gower St, London WC1E 6BT, UK +Protein and Peptide Group, EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany §The Ludwig Institute for Cancer Research, 91 Riding House Street London W1P 8BT, UK Cell, in press. Summary The TTGA binding factor is a transcriptional regulator activated by DIF, the chlorinated hexaphenone that induces prestalk cell differentiation in Dictyostelium . The same activity also functions as a repressor, controlling stalk cell differentiation. We show that the TTGA binding factor is a STAT protein. Like the metazoan STATs, it functions via the reciprocal interaction of a phosphotyrosine residue on one molecule with an SH2 domain on a dimerising partner. Also, it will bind specifically to a mammalian alpha interferon response element. In Saccharomyces cerevisiae, where the entire genomic sequence is known, SH2 domains have not been identified. It would seem therefore that SH2 signalling pathways arose very early in the evolution of multicellular organisms, perhaps to facilitate intercellular comunication. --------------------------------------------------------------------- CudA: a Dictyostelium gene with pleiotropic effects on cellular differentiation and slug behaviour Masashi Fukuzawa, Neil Hopper and Jeffrey Williams+ MRC Laboratory of Molecular Cell Biology and Department of Biology, University College London, Gower St, London WC1E 6BT +Author for correspondence (e-mail jeff.williams@ucl.ac.uk) Development, in press. Summary The Dictyostelium cudA gene encodes a nucleoplasmic protein that is essential for normal culmination. There are no functionally characterised homologues in other organisms but there is a related gene of unknown function in Entamoeba histolytica. The cudA gene is expressed by the prestalk cells that constitute the slug tip (the pstA cells), it is not detectably expressed in the band of prestalk cells that lies behind the tip (the pstO cells) but it is expressed in the prespore cells. This unusual pattern of expression suggests a role on both the stalk and spore pathways of differentiation and cudA- mutant cells are indeed defective in both stalk and spore formation. Furthermore, the slugs formed by cudA- cells continue to migrate under environmental conditions where normal slugs culminate immediately. This aspect of their behaviour can be reversed when the cudA gene is selectively expressed in the pstA cells. This shows that processes occuring in the pstA cells regulate entry into culmination. --------------------------------------------------------------------- X-ray analysis of AZT diphosphate : Binding to Nucleoside Diphosphate Kinase Yingwu Xu, Olivier Sellam, Solange Moréra, Simon Sarfati, Ricardo Biondi, Michel Véron and Joël Janin Laboratoire d'Enzymologie et de Biochimie Structurales, UPR 9063 CNRS 91198-Gif-sur-Yvette, France and Unité de Régulation Enzymatique des Activités Cellulaires, Institut Pasteur, 75724-Paris Cedex 15, France Proc. Natl. Acad. Sci., in press ABSTRACT AZT and the dideoxy- analogs of cytosine and inosine are major drugs against retroviral diseases. Upon infection by human immunodeficiency virus, these nucleoside analogs are incorporated by reverse transcriptase copying the viral genome, and DNA synthesis terminates, for they lack the 3'OH needed for elongation of the polynucleotide chain. Since substrates for reverse transcriptase are (deoxy)nucleoside triphosphates, the analogs must undergo phosphorylation before incorporation. This reaction is performed in several steps by cellular phosphokinases and it is very slow beyond the first step. Thus, lymphocytes incubated with AZT accumulate inactive intermediates . With natural nucleotides, the conversion of the di- to the triphosphate is efficiently carried out by nucleoside diphosphate (NDP) kinase, an enzyme present in all organisms. Human cells have two isotypes, NDK kinases A and B, the products of the nm23-H1 and nm23-H2 genes. When the capacity of human NDP kinase B to phosphorylate derivatives of AZT and dideoxy-nucleosides has been tested, it was found to be several orders of magnitude less than for natural substrates. This may make triphosphate production rate-limiting in HIV inhibition by the drugs, and suggests that analogs that are better substrates of NDP kinase should also be better drugs. To understand why NDP kinase is so unefficient in activating AZT, we determined a 2.3 Å X-ray structure of a complex with AZT diphosphate (AZT-DP) and compared it with a previously determined complex with thymidine diphosphate (dTDP). The enzyme was a point mutant of the NDP kinase of the slime mold Dictyostelium discoideum, a 100 kDa hexamer which is highly homologous to the human enzyme (57% sequence identity), and has a very similar three-dimensional structure, especially at the active site. The structure of the complex shows that the analog binds at the same site and in the same way as natural substrates. It brings strong support to the conclusion that the 3'OH group of the sugar, missing in dideoxy compounds and replaced with an azido group in AZT, is an essential component of the mechanism of phosphate transfer catalysis by NDP kinase. --------------------------------------------------------------------- [End CSM News, volume 8, number 11]