Dicty News Electronic Edition Volume 20, number 2 February 15, 2003 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ============= Abstracts ============= DIF-1, an anti-tumor substance found in Dictyostelium discoideum, inhibits progesterone-induced oocyte maturation in Xenopus laevis Yuzuru Kuboharaa*, Yoichi Hanaoka, Emi Akaishi, Hisae Kobayashi, Mineko Maeda, Kohei Hosaka Biosignal Research Center, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371-8512, Japan European Journal of Pharmacology, In press Abstract Differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy- 4-methoxyphenyl)hexan-1-one) is a putative morphogen that induces stalk- cell formation in the cellular slime mold Dictyostelium discoideum. DIF-1 has previously been shown to suppress cell growth in mammalian cells. In this study, we examined the effects of DIF-1 on the progesterone-induced germinal vesicle breakdown in Xenopus laevis, which is thought to be mediated by a decrease in intracellular cAMP and the subsequent activation of mitogen-activated protein kinase (MAPK) and maturation-promoting factor, a complex of cdc2 and cyclin B, which regulates germinal vesicle breakdown. DIF-1 at 10-40 micro M inhibited progesterone-induced germinal vesicle breakdown in de-folliculated oocytes in a dose-dependent manner. Progesterone-induced cdc2 activation, MAPK activation, and c-Mos accumulation were inhibited by DIF-1. Furthermore, DIF-1 was found to inhibit the progesterone-induced cAMP decrease in the oocytes. These results indicate that DIF-1 inhibits progesterone-induced germinal vesicle breakdown possibly by blocking the progesterone-induced decrease in [cAMP]i and the subsequent events in Xenopus oocytes. ---------------------------------------------------------------------------- The AP-1 clathrin-adaptor is required for lysosomal enzymes sorting and biogenesis of the contractile vacuole complex in Dictyostelium cells Yaya Lefkir,* Benot de Chassey,* Annick Dubois,* Aleksandra Bogdanovic, Rebecca J. Brady, Olivier Destaing, Franz bruckert, Theresa J. O'halloran, Pierre Cosson, and Franois Letourneur*# * Institut de Biologie et Chimie des Protines, UMR5086 - CNRS/Universit Lyon I 7, Passage du Vercors, 69367 Lyon cedex 07, France; Laboratoire de Biochimie et Biophysique des Systmes Intgrs, 38054 Grenoble Cedex 9, France; 241 Patterson Laboratories, Section of Molecular Cell & Developmental Biology, The University of Texas at Austin, 24th St. and Speedway Avenue, Austin, TX 78712, USA; Laboratoire de Biologie Molculaire et Cellulaire / UMR 5665 Ecole Normale Suprieure de Lyon, 69364 LYON Cedex 07, France; Universit de Genve, Centre Mdical Universitaire, Dpartement de Morphologie, CH-1211 Genve 4, Switzerland Molecular Biology of the Cell, In Press ABSTRACT Adaptor protein complexes (AP) are major components of the cytoplasmic coat found on clathrin-coated vesicles. Here, we report the molecular and functional characterization of Dictyostelium clathrin-associated AP-1 complex, which in mammalian cells, participates mainly in budding of clathrin-coated vesicles from the trans-Golgi network (TGN). The g-adaptin AP-1 subunit was cloned and shown to belong to a Golgi-localized 300kDa protein complex. Time-lapse analysis of cells expressing g-adaptin tagged with the green-fluorescent-protein demonstrates the dynamics of AP-1 coated structures leaving the Golgi apparatus and rarely moving towards the TGN. Targeted disruption of the AP-1 medium chain results in viable cells displaying a severe growth defect and a delayed developmental cycle as compared to parental cells. Lysosomal enzymes are constitutively secreted as precursors, suggesting that protein transport between the TGN and lysosomes is defective. Whereas endocytic protein markers are correctly localized to endosomal compartments, morphological and ultrastructural studies reveal the absence of large endosomal vacuoles and an increased number of small vacuoles. In addition, the function of the contractile vacuole complex (CV), an osmoregulatory organelle is impaired and some CV components are not correctly targeted. submitted by: Francois Letourneur [f.letourneur@ibcp.fr] ---------------------------------------------------------------------------- Title: Analysis of 5 Nucleotidase and Alkaline Phosphatase by Gene Disruption in Dictyostelium Charles L. Rutherford*, Danielle F. Overall, Muatasem Ubeidat and Bradley R. Joyce Biology Department, Molecular and Cellular Biology Section, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406, USA Genesis, In Press Abstract In Dictyostelium discoideum a phosphatase with a high pH optimum is known to increase in activity during cell differentiation and become localized to a narrow band of cells at the interface of prespore and prestalk cells. However, it was not clear if this activity is due to a classical alkaline phosphatase with broad range substrate specificity or to a 5 nucleotidase with high substrate preference for 5 AMP. We attempted to disrupt the genes encoding these two phosphatase activities in order to determine if the activity that is localized to the interface region resides in either of these two proteins. During aggregation of 5nt null mutants multiple tips formed rather than the normal single tip for each aggregate. In situ phosphatase activity assays showed that the wt and the 5nt gene disruption clones had normal phosphatase activity in the area between prestalk and prespore cell types, while the alp null mutants did not have activity in this cellular region. Thus, the phosphatase activity that becomes localized to the interface of the prestalk and prespore cells is Alkaline Phosphatase. Submitted by rutherfo@vt.edu ---------------------------------------------------------------------------- ADENYLYL CYCLASE LOCALIZATION REGULATES STREAMING DURING CHEMOTAXIS Paul W. Kriebel, Valarie A. Barr, and Carole A. Parent* Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health Cell, In press. We studied the role of the adenylyl cyclase ACA in D. discoideum chemotaxis and streaming. In this process cells orient themselves in a head to tail fashion as they are migrating to form aggregates. We show that cells lacking ACA are capable of moving up a chemoattractant gradient, but are unable to stream. Imaging of ACA-YFP reveals plasma membrane labeling highly enriched at the uropod of polarized cells. This localization requires the actin cytoskeleton but is independent of the regulator CRAC and the effector PKA. A constitutively active mutant of ACA shows dramatically reduced uropod enrichment and has severe streaming defects. We propose that the asymmetric distribution of ACA provides a compartment from which cAMP is secreted to locally act as a chemoattractant, thereby providing a unique mechanism to amplify chemical gradients. This could represent a general mechanism that cells use to amplify chemotactic responses. submitted by Carole Parent [parentc@helix.nih.gov] ---------------------------------------------------------------------------- Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum Marcel E. Meima, Karin E. Weening and Pauline Schaap School of Life Sciences, University of Dundee, MSI/WTB complex, Dow Street, Dundee DD1 5EH, UK. SUMMARY A cyclic nucleotide phosphodiesterase, PdeE, that harbours two cyclic nucleotide binding motifs and a binuclear Zn2+ binding domain, was characterized in Dictyostelium. In other eukaryotes, the Dictyostelium domain shows greatest homology to the 73 kD subunit of the premRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at highest levels during aggregation and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-YFP1 fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was partially purified by immuno-precipitation and biochemically characterized. PdeE and its Dictyostelium ortholog PdeD are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn2+ and Mg2+ activated PdeE and PdeD maximally at 10 mM, while Mn2+ activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 M. PdeE is an allosteric enzyme, which is about 4-fold activated by cAMP, with half-maximal activation occurring at about 10 M and an apparent KM around 1 mM. cGMP is degraded at a 6-fold lower rate than cAMP. Neither cGMP nor 8BrcAMP are efficient activators of PdeE activity. submitted by Pauline Schaap [p.schaap@dundee.ac.uk] ---------------------------------------------------------------------------- Differentiation-inducing factor-1 (DIF-1) inhibits STAT3 activity involved in gastric cancer cell proliferation via MEK-ERK-dependent pathway. Kanai M, Konda Y, Nakajima T, Izumi Y, Kanda N, Nanakin A, Kubohara Y, Chiba T. Kyoto University School of Medicine, Gunma University IMCR Oncogene (2003) 22, 548-554. Differentiation-inducing factor-1 (DIF-1) is a chlorinated hexaphenone isolated from Dictyostelium. DIF-1 exhibits antitumor activity in several types of mammalian tumor cells, although the underlying mechanisms remain unknown. On the other hand, recent studies indicate that constitutively activated STAT3 acts as an oncogene and could be a target for antitumor drug. In the present study, we examined the effects of DIF-1 on proliferation of gastric cancer cell lines as well as on its signal transduction pathways, focusing mainly on STAT proteins. DIF-1 inhibited proliferation of gastric cancer cells. Western blot analysis and electrophoretic mobility shift assay showed that DIF-1 inhibited STAT3 activity in an MEK-ERK-dependent manner in gastric cancer cell lines, AGS and MKN28. Moreover, blockade of STAT3 activity by ectopic expression of dominant-negative STAT3 or the Janus kinase inhibitor, tyrphostin AG490, inhibited cell growth of AGS cells. These results suggest that STAT3 activity plays an important role for cell growth in AGS cells, and raises the possibility that inhibition of STAT3 activity is one of the mechanisms responsible for the antitumor effect of DIF-1 in these cells. ---------------------------------------------------------------------------- [End Dicty News, volume 20, number 2]