dictyNews Electronic Edition Volume 26, number 13 May 5, 2006 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= EppA, a putative substrate of DdERK2, regulates cAMP relay and chemotaxis in Dictyostelium discoideum Songyang Chen and Jeffrey E. Segall* Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 Eukaryotic Cell, in press The MAP kinase DdERK2 is critical for cAMP relay and chemotaxis to cAMP and folate, but the details downstream of DdERK2 are unclear. To search for targets of DdERK2 in Dictyostelium, 32PO43--labeled protein samples from wild type and Dderk2- cells were resolved by 2 dimensional electrophoresis. Mass spectrometry was used to identify a novel 45kD protein as a substrate of DdERK2 in Dictyostelium named EppA (ERK2-dependent phosphoprotein A). Mutation of potential DdERK2 phosphorylation sites demonstrated that phosphorylation on serine 250 of EppA is DdERK2-dependent. Changing serine 250 to alanine delayed development of Dictyostelium and reduced Dictyostelium chemotaxis to cAMP. Although overexpression of EppA had no significant effect on development or chemotaxis of Dictyostelium, disruption of the eppA gene led to delayed development and reduced chemotactic responses to both cAMP and folate. Both eppA gene disruption and overexpression of EppA carrying the serine 250 to alanine mutation led to inhibition of intracellular cAMP accumulation in response to chemoattractant cAMP, a pivotal process in Dictyostelium chemotaxis and development. Our studies indicate that EppA regulates extracellular cAMP induced signal relay and chemotaxis of Dictyostelium. Submitted by: Jeffrey E. Segall [segall@aecom.yu.edu] ----------------------------------------------------------------------------- A cis-acting site controlling bidirectional transcription at the growth-differentiation transition in Dictyostelium Shigenori Hirose, Samuel H. Payne and William F. Loomis Cell and Developmetal Biology, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0368, USA Eukary. Cell, in press A pair of adjacent genes, impA and dia1, are divergently transcribed but expressed at different stages in the life cycle of Dictyostelium discoideum. The intervening 654 bp region carries cis-acting regions that are essential for transcription in both directions as well as repression of dia1 in growing cells. We have focused on a 112 bp region proximal to dia1 that is essential for bidirectional transcription. Analyses of a set of internal deletions showed that the sequence between positions 80 and 97 (TTTGAATTTTTTGAATTT) is critical and that bases outside this region are dispensable. Site directed mutations within this critical region confirmed the importance of this sequence for transcription both to the right and to the left. However, insertions of either 6 or 24 Ts into the run of 6 Ts separating the repeated GAA sequence had little effect on the functioning of the site in either direction suggesting that factors recognize the half sites TTGAATT separately. Inversion of the bases between positions 80 and 97 greatly reduced expression in both directions indicating that orientation is critical for expression of both the near-by impA gene and the distal dia1 gene which is more than 500 bp away. Comparison of 38 mutant constructs with multiple random variations in the region indicated that transcription factors may bind to a range of related sequences and still retain function. All functional constructs directed transcription both leftward and rightward while all non-functional constructs were impaired for transcription in both directions. It appears that the same transcription complex controls transcription of both impA and dia1. Submitted by: Bill Loomis [wloomis@ucsd.edu] ----------------------------------------------------------------------------- Filamins: promiscuous organizers of the cytoskeleton Grzegorz M. Popowicz1, Michael Schleicher2, Angelika Noegel3, and Tad A. Holak1 1 Max-Planck Institut fŸr Biochemie, 82152 Martinsried, Germany 2 Institut fŸr Zellbiologie der LMU, 80336 MŸnchen, Germany 3 Institut fŸr Biochemie I, Zentrum Molekulare Medizin Kšln, Medizinische FakultŠt, Univ. zu Kšln, 50931 Kšln, Germany Trends in Biochemical Sciences, in press Filamins are F actin cross-linking proteins. They are elongated homodimers; each monomer chain comprises an actin-binding domain and a rod segment which consists of six (Dictyostelium filamin) up to 24 (human filamin) highly homologous repeats of 96 amino acid residues which adopt an immunoglobulin-like fold. Hinges in the rod segment and reversible unfolding of single repeats might be the structural basis for the intrinsic flexibility of the actin networks generated by filamins. There is a plethora of filamin binding proteins which in most cases bind along the rod repeats. This rather promiscuous behaviour renders a filamin a versatile scaffold between the actin network and finely tuned molecular cascades from the membrane to the cytoskeleton. Submitted by: Paul Steimle [noegel@uni-koeln.de] ----------------------------------------------------------------------------- The function of ammonium transporter A in the initiation of culmination of development in Dictyostelium discoideum Charles K. Singleton*, Janet H. Kirsten, and Colin J. Dinsmore Department of Biological Sciences Vanderbilt University VU Station B 351634 Nashville TN 37235-1634, USA Eukaryotic Cell, in press The histidine kinase DhkC controls a phosphorelay involved in regulating the slug versus culmination choice during the multicellular developmental program of Dictyostelium discoideum. When the relay is active, slug migration is favored due to the activation of a cAMP phosphodiesterase and the resultant lowering of the intracellular and extracellular levels of cAMP. Ammonia signaling represents one input into the DhkC phosphorelay, and previous studies indicated that the ammonium transporter C inhibits the relay in response to low ammonia levels. Evidence is presented that another member of the family of ammonium transporters, AmtA, also regulates the slug/culmination choice. Under standard conditions of development, the wild type strain requires a transitional period of 2 to 3 hours to go from fingers to culminants, with some slugs forming and migrating briefly prior to culmination. In contrast, amtA null cells, like cells that lack DhkC, possessed a transitional period of only 1 to 2 hours and rarely formed slugs. Disruption of amtA in an amtC null strain overcame the slugger phenotype of that strain and restored its ability to culminate. Strains lacking AmtA were insensitive to the ability of ammonia to promote and prolong slug migration. These findings lead to the proposal that AmtA functions in ammonia sensing as an activator of the DhkC phosphorelay in response to perceived high ammonia levels. Submitted by: Charles Singleton [charles.k.singleton@vanderbilt.edu] ----------------------------------------------------------------------------- DdPDE4, a novel cAMP-specific Phosphodiesterase at the Surface of Dictyostelium Cells Sonya Bader, Arjan Kortholt*, Helena Snippe and Peter J.M. Van Haastert Department of Molecular Cell Biology, University of Groningen, Kerklaan 30, 9751NN Haren, the Netherlands Journal of Biological Chemistry, in press Dictyostelium discoideum cells possess multiple cyclic nucleotide phosphodiesterases that belong either to class I enzymes that are present in all eukaryotes, or to the rare beta-lactamase class II. We describe here the identification and characterization of DdPDE4, the third class I enzyme of Dictyostelium. The deduced amino acid sequence predicts that DdPDE4 has a leader sequence, two transmembrane segments and an extracellular catalytic domain that exhibits a high degree of homology with human cAMP-specific PDE8. Expression of the catalytic domain of DdPDE4 shows that the enzyme is a cAMP-specific phosphodiesterase activity with a KM of 10 “M; cGMP is hydrolyzed at least 100-fold more slowly. The full length protein is shown to be membrane-bound with catalytic activity exposed to the extracellular medium. Northern blots and activity measurements reveal that expression of DdPDE4 is low during single cell stages, and increases at nine hours of starvation, corresponding with mound stage. A function during multicellular development is confirmed by the phenotype of ddpde4- knock-out strains, showing normal aggregation but impaired development from the mound stage on. These results demonstrate that DdPDE4 is a unique membrane-bound phosphodiesterase with an extracellular catalytic domain regulating inter-cellular cAMP during multicellular development. Submitted by: Peter Van Haastert [p.j.m.van.haastert@rug.nl] ============================================================================== [End dictyNews, volume 26, number 13]