Dicty News Electronic Edition Volume 10, number 10 April 11, 1998 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" ========================= Franke Database Updated ========================= An updated version of the Franke Dictyostelium reference database is now available through the Dicty web page at http://dicty.cmb.nwu.edu/dicty/dicty.html =========== Abstracts =========== CHARACTERIZATION AND CLONING OF A DICTYOSTELIUM STE20-LIKE PROTEIN KINASE THAT PHOSPHORYLATES THE ACTIN-BINDING PROTEIN SEVERIN Ludwig Eichinger1, Martin Bähler1, Melanie Dietz1, Christoph Eckerskorn2, and Michael Schleicher1 1Adolf-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universität, Schillerstr. 42, 80336 München, Germany; 2Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany J. Biol. Chem., in press Abstract After receiving an external stimulus Dictyostelium amoebae are able to rearrange their actin cytoskeleton within seconds, and phosphorylation is a prime candidate for quick modification of cytoskeletal components. We isolated a kinase from cytosolic extracts that specifically phosphorylated severin, a Ca2+-dependent F-actin fragmenting protein. In gel filtration chromatography severin kinase eluted with a molecular mass of about 300 kDa and contained a 62 kDa component whose autophosphorylation caused a mobility shift in SDS-PAGE and stimulated phosphorylation of severin. Severin kinase activity could be specifically precipitated with antibodies raised against the 62 kDa polypeptide. Phosphorylation of severin was strongly reduced in the presence of Ca2+, indicating additional regulation at the substrate level. Peptide sequencing and cloning of the cDNA demonstrated that the 62 kDa protein belongs to the Ste20p- or p21-activated protein kinase family. It is most closely related to the germinal center kinase subfamily with its N-terminally positioned catalytic domain followed by a presumptive regulatory domain at the C-terminus. The presence of a Ste20-like severin kinase in Dictyostelium suggests a direct signal transduction from the plasma membrane to the cytoskeleton by phosphorylation of actin-binding proteins. ------------------------------------------------------------------------- Diphospho-myo-inositolphosphates in Dictyostelium and Polysphondylium: Identification of a new bisdiphospho-myo-inositol tetrakisphosphate Tim Laussmann1, André Hansen1, Komandla Malla Reddy2, K. Kishta Reddy2, J.R. Falck2 and Guenter Vogel1* 1Fachbereich 9 - Chemie, Bergische Universitaet GHS Wuppertal, Gaussstrasse 20, 42097 Wuppertal. 2Department of Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9038 Abstract The two major diphospho inositol phosphates from the axenic strain D. discoideum AX2 were previously investigated and identified as 6-PP-InsP5 and 5,6-bis-PP-InsP4. In order to examine whether these findings are representative of Dictyostelids in general, five non- axenic wild type species of Dictyostelium and two of Polysphondylium were studied. It was found that all of the Dictyostelium species exhibit similar patterns of diphospho inositol phosphates. By contrast, both of the Polysphondylium species contain 5-PP-InsP5 as the predominant isomer. Besides 5,6-bis-PP-InsP4, a new bis-PP-InsP4 was detected in Polysphondylium. This compound is either 1,5-bis-PP-InsP4 or its corresponding enantiomer 3,5-bis-PP-InsP5. The structures were elucidated by two dimensional 1H-1H and 1H-31P NMR analysis. Additionally, they were confirmed using a specific 6-PP-InsP5-5-kinase from D. discoideum AX2 as an enantiospecific tool and enantiomerically pure reference standards. ------------------------------------------------------------------------- The S. cerevisiae CLU1 and D. discoideum cluA genes are functional homologues that influence mitochondrial morphology and distribution. Stephen D. Fields, Michael N. Conrad, and Margaret Clarke Oklahoma Medical Research Foundation 825 N.E. 13th Street Oklahoma City, OK 73104 J. Cell Sci., in press Summary The cluA gene, encoding a novel 150-kDa protein, was recently characterized in Dictyostelium discoideum; disruption of cluA impaired cytokinesis and caused mitochondria to cluster at the cell center (Zhu et al., Proc. Natl. Acad. Sci. USA 94, 7308-7313, 1997). The genome of Saccharomyces cerevisiae contains an open reading frame (CLU1) that encodes a protein that is 27% identical, 50% similar, to this Dictyostelium protein. Deletion of CLU1 from S. cerevisiae did not affect cell viability, growth properties, sporulation efficiency, or frequency of occurrence of cells lacking functional mitochondria. However, in clu1D cells the mitochondrial reticulum, which is normally highly branched, was condensed to one side of the cell. Transformation of cluA- Dictyostelium mutants with the yeast CLU1 gene yielded amoebae that divided normally and had dispersed mitochondria. The mitochondria in cluA- Dictyostelium cells complemented with CLU1 were not as widely scattered as in cluA+ Dictyostelium cells, but formed loose clusters throughout the cytoplasm. These results indicate that the products of the CLU1 and cluA genes, in spite of their limited homology, are functional homologues. ------------------------------------------------------------------------- YakA, a protein kinase required for the growth to development transition inDictyostelium Glaucia Mendes Souza, Sijie Lu and Adam Kuspa Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas, USA, 77030, Dept. Bioquimica, Instituto de Quimica, University of Sao Paulo, Brazil 05508 Development, in press SUMMARY When Dictyostelium cells starve they arrest their growth and induce the expression of genes necessary for development. We have identified and characterized a protein kinase, YakA, that is essential for the proper regulation of both events. Amino acid sequence and functional similarities indicate that YakA is a homolog of Yak1p, a growth-regulating protein kinase in S. cerevisiae. Purified YakA expressed in E. coli is able to phosphorylate myelin basic protein. YakA-null cells are smaller and their cell cycle is accelerated relative to wild-type cells. When starved, YakA-null cells fail to decrease the expression of the growth-stage gene cprD, and do not induce the expression of genes required for the earliest stages of development. YakA mRNA levels increase during exponential growth and reach a maximum at the point of starvation, consistent with a role in mediating starvation responses. YakA mRNA also accumulates when cells are grown in media conditioned by cells grown to high density suggesting that yakA expression is under the control of an extracellular signal that accumulates during growth. Expression of yakA from a conditional promoter causes cell cycle arrest in nutrient- rich medium and promotes developmental events, such as the expression of genes required for cAMP signaling. YakA appears to regulate the transition from growth to development in Dictyostelium. ------------------------------------------------------------------------- [End Dicty News, volume 10, number 10]