Dicty News Electronic Edition Volume 11, number 1 July 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" =========== Abstracts =========== Inhibition of the cAMP dependent protein kinase by synthetic A-helix peptides Stéphane Gamboni, Catherine Chaperon, Kirstin Friedrich§, Pascal J. Baehler, Christophe D. Reymond Insitut de biologie cellulaire et de morphologie, Rue du Bugnon 9, CH-1005 Lausanne, Switzerland § EPFL, Laboratoire de chimie physique des polymères et membranes, CH-1015 Lausanne. BIOCHEMISTRY (In Press) Abstract: The catalytic subunit of the cAMP-dependent protein kinase from Dictyostelium discoideum, PkaC, displays the same properties as its mammalian counterpart, except for being about twice as large in size. Sequence comparisons indicated the presence of a conserved a-helix (A-helix) within the N-terminal region of PkaC which could potentially establish close contacts with the catalytic core (1). We show in this report that a synthetic peptide with the A-helix sequence inhibits PKA activity, whereas unrelated peptides display no inhibitory activity. The inhibition seems competitive with respect to the kemptide substrate rather than due to binding to a secondary site. We further show by amino acid replacements that the last lysine of the A-helix sequence is involved in this specific inhibition. A model is proposed for the possible role of the A-helix. ------------------------------------------------------------------------- Interaptin, an actin-binding protein of the a-actinin superfamily in Dictyostelium discoideum, is developmentally and cAMP-regulated and associates with intracellular membrane compartments Francisco Rivero1,2, Adam Kuspa3, Regine Brokamp1, Monika Matzner1 and Angelika A. Noegel1,2,4 1Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany. 2Institut für Biochemie I, Medizinische Fakultät, Universität zu Köln, Joseph-Stelzmann-Str. 52, 50931 Köln, Germany 3Department of Biochemistry, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030 Journal of Cell Biology, in press. In a search for novel members of the a-actinin superfamily a D. discoideum genomic library in YACs was screened under low stringency conditions using the acting-binding domain of the gelation factor as probe. A new locus was identified and 8.6 kb of genomic DNA were sequenced that encompassed the whole abpD gene. The DNA sequence predicts a protein, interaptin, with a calculated molecular mass of 204,300 that is constituted by an actin-binding domain, a central coiled-coil rod domain and a membrane-associated domain. In Northern blot analyses a cAMP-stimulated transcript of 5.8 kb is expressed at the stage when cell differentiation occurs. Monoclonal antibodies raised against bacterially expressed interaptin polypeptides recognized a 200 kD developmentally and cAMP-regulated protein and a 160 kD constitutively expressed protein in Western blots. In multicellular structures interaptin appears to be enriched in anterior-like cells which during culmination sort to the upper and lower cups. The protein is located at the nuclear envelope and ER. In mutants deficient in interaptin development is delayed, but the morphology of the mature fruiting bodies appears normal. When starved in suspension abpD- cells form EDTA-stable aggregates, which, in contrast to wild type, dissociate. Based on its domains and location interaptin constitutes a potential link between intracellular membrane compartments and the actin cytoskeleton. ------------------------------------------------------------------------- Kinetic Characterization of Myosin Head Fragments with Long-lived Myosin.ATP States A. L. Friedman(1), M. A. Geeves(2), D. J. Manstein(3), and J. A. Spudich*(1) Department of Biochemistry, Stanford University, Stanford, California, 94305, Max-Planck-Institut fur Molekulare Physiologie, Postfach 102644, D-44026 Dortmund, and Max-Planck-Institut fur Medizinische Forschung, Jahnstrasse 29, D-69120 Heidelberg, Germany Biochemistry, in press. ABSTRACT: We have separately expressed the D. discoideum myosin II nonhydrolyzer point mutations E459V and E476K [Ruppel, K. M. and Spudich, J. A. (1996) Mol. Biol. Cell 7, 1123-1136.] in the soluble myosin head fragment M761-1R [Anson et al. (1996) EMBO J. 15, 6069-6074] and performed transient kinetic analyses to characterize the ATPase cycles of the mutant proteins. While the mutations cause some changes in mantATP (2’(3’)-O-(N-methylanthraniloyl) -ATP) and mantADP binding, the most dramatic effect is on the hydrolysis step of the ATPase cycle, which is reduced by four (E476K) and six (E459V) orders of magnitude. Thus both mutant myosin constructs do in fact catalyze ATP hydrolysis but have very long-lived myosin.ATP states. The E459V mutation allowed for a direct measurement of the ATP off rate constant from myosin, which was found to be 2 x 10-5 s-1. Actin accelerated ATP release from this E459V construct by at least 100-fold. Additionally, we found that the affinity of the E476K construct for actin is significantly weaker than for the wild-type construct, while the E459V mutant interacts with actin normally. Their functional properties and the fact that they can be produced and purified in large amounts makes the E476K and E459V constructs ideal tools to elucidate key structural features of the myosin ATPase cycle. These constructs should allow us to address important questions, including how binding of ATP to myosin heads results in a >3 order of magnitude reduction in actin affinity. ------------------------------------------------------------------------- A Novel Component Involved in Ubiquitination is Required for Development of Dictyostelium discoideum Stefan Pukatzki, Nelson Tordilla, Jakob Franke, and Richard H. Kessin Department of Anatomy and Cell Biology, College of Physicians & Surgeons, Columbia University, New York, NY 10032, U.S.A. Journal of Biological Chemistry. In Press. Abstract A novel component of the ubiquitination system, called NOSA, is essential for cellular differentiation in Dictyostelium discoideum. Disruption of nosA does not affect the growth rate but causes an arrest in development after the cells have aggregated. NosA contains seven exons, and codes for a develop- mentally regulated 3.5-kb mRNA. The 125-kD NOSA protein is present in the cytosol at constant levels during growth and development. The C-terminal region of NOSA has homology with UFD2 of Saccharomyces cerevisiae and putative homologs in Caenorhabditis elegans and humans. UFD2 is involved in the ubiquitin-mediated degradation of model substrates in which ubiquitin forms part of the translation product, but ufd2 mutants have no detected phenotype. In accord with the homology to UFD2, we found differences in ubiquitination pattern between nosA mutants and their parental cell line. While general in vivo and in vitro ubiquitination is minimally affected, ubiquitination of individual proteins is altered throughout growth and development in nosA mutants. These findings suggest that events involving ubiquitination are critical for progression through the aggregate stage of the Dictyostelium life cycle. ------------------------------------------------------------------------- Temperature-sensitive Gbeta mutants Discriminate Between G-protein Dependent and G-protein Independent Signaling Mediated by Serpentine Receptors. Tian Jin1, Ron D. M. Soede2, Jingchun Liu3, Alan R. Kimmel3, Peter N. Devreotes1 and Pauline Schaap2* 1Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205. 2Cell Biology Section, Institute for Molecular Plant Sciences, University of Leiden, The Netherlands. 3Laboratory of Cell and Developmental Biology, National Institutes of Health, Bethesda, Maryland 20892. EMBO J. in press ABSTRACT Deletion of the single gene for the Dictyostelium G-protein beta-subunit blocks development at an early stage. We have now isolated temperature-sensitive alleles of G-beta to investigate its role in later development. We show that G-beta is directly required for adenylyl cyclase A activation and for morphogenetic signaling during the entire developmental program. G-beta was also essential for induction of aggregative gene expression by cAMP pulses, a process that is mediated by serpentine cAMP receptors (cARs). However, G-beta was not required for cAR-mediated induction of prespore genes and repression of stalk genes and neither was G-beta needed for induction of prestalk genes by the stalk-inducing morphogen, DIF. cAMP induction of prespore genes and repression of stalk genes is mediated by the protein kinase GSK-3. GSK-3 also determines cell-type specification in insects and vertebrates and is regulated by the wingless/wnt morphogens that are detected by serpentine fz receptors. The G-protein dependent and independent modes of cAR-mediated signaling reported here may also exist for the wingless/wnt signaling pathways in higher organisms. ------------------------------------------------------------------------- FUNCTIONAL PROMISCUITY OF GENE REGULATION BY SERPENTINE RECEPTORS IN DICTYOSTELIUM Irene Verkerke-Van Wijk1, Ji-Yun Kim2, Raymond Brandt1, Peter N. Devreotes2 and Pauline Schaap1* 1.Cell Biology Section, Institute for Molecular Plant Sciences, University of Leiden, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. 2.Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Molecular and Cellular Biology in press. SUMMARY Serpentine receptors as smoothened and frizzled play important roles in cell fate determination during animal development. In Dictyostelium, four serpentine cAMP receptors (cARs) regulate expression of multiple classes of developmental genes. To understand their function, it is essential to know whether each cAR is coupled to a specific gene regulatory pathway or whether specificity results from the different developmental regulation of individual cARs. To distinguish between these possibilities, we measured gene induction in car1-/car3- cell lines, that express equal levels of either cAR1, cAR2 or cAR3 under a constitutive promoter. We found that all cARs efficiently mediate both aggregative gene induction by cAMP pulses as well as induction of postaggregative and prespore genes by persistent cAMP stimulation. Two exceptions to this functional promiscuity were observed: i. Only cAR1 can mediate adenosine inhibition of cAMP-induced prespore gene expression, a phenomenon that was found earlier in wild-type cells. This suggest that cAR1 normally mediates prespore gene induction. ii. Only cAR2 allows entry into the prestalk pathway. Prestalk gene expression is induced by the lipophilic signal DIF, but only after cells have been prestimulated with cAMP. We found that DIF-induced prestalk gene expression is 10 times higher in constitutive cAR2 expressors than in constitutive cAR1 or cAR3 expressors (which still have endogenous cAR2), suggesting that cAR2 mediates induction of DIF competence. Since in wild-type slugs, cAR2 is only expressed in anterior cells, this could explain the thusfar puzzling observation that prestalk cells differentiate at the anterior, while DIF levels are higher at the posterior region. After the initial induction of DIF competence, cAMP becomes a repressor of prestalk gene expression. This function can again be mediated by cAR1, cAR2 and cAR3. ------------------------------------------------------------------------- Cell Type Specific Rescue of Myosin Function During Dictyostelium Development Defines Two Distinct Cell Movements Required for Culmination Tung-Ling L. Chen, Wendy A. Wolf and Rex L. Chisholm Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611 Development, in press. SUMMARY Mutant Dictyostelium cells lacking any of the component polypeptides of myosin II exhibit developmental defects. To define myosin's role in establishing Dictyostelium’s developmental pattern, we have rescued myosin function in a myosin regulatory light chain null mutant (mlcR-) using cell type specific promoters. While mlcR- cells fail to progress beyond the mound stage, expression of RLC from the prestalk promoter, ecmA, produces culminants with normal stalks but with defects in spore cell localization. When GFP marked prestalk and prespore cells expressing ecmA-RLC are mixed with wildtype cells, the mislocalization of prestalk cells, but not prespore cells, is rescued. Time-lapse video recording of ecmA-RLC cells showed that the posterior prespore zone failed to undergo a contraction important for the upward movement of prespore cells. Prespore cells marked with green fluorescent protein (GFP) failed to move toward the tip with the spiral motion typical of wildtype. In contrast, expression of RLC in prespore cells using the psA promoter produced balloon-like structures reminiscent of sorocarps but lacking stalks. GFP-labeled prespore cells showed a spiral movement toward the top of the structures. Expression of RLC from the psA promoter restores the normal localization of psA-GFP cells, but not ecmA-GFP cells. These results define two distinct, myosin-dependent movements that are required for establishing a Dictyostelium fruiting body: stalk extension and active movement of the prespore zone that ensures proper placement of the spores atop the stalk. The approach used in these studies provides a direct means of testing the role of cell motility in distinct cell types during a morphogenetic program. ------------------------------------------------------------------------- [End Dicty News, volume 11, number 1]