CSM News Electronic Edition Volume 7, number 15 December 14, 1996 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 by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== Substitution Mutations in the Myosin Essential Light Chain Lead to Reduced Actin-activated ATPase Activity Despite Stoichiometric Binding to the Heavy Chain Guyu Ho and Rex L. Chisholm* Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611 J. Biol. Chem., in press. Myosin essential light chain (ELC) wraps around an alpha-helix that extends from the myosin head, where it is believed to play a structural support role. To identify other role(s) of the ELC in myosin function, we have used an alanine scanning mutagenesis approach to convert charged residues in loops I, II, III, and helix G of the Dictyostelium ELC into uncharged alanines. Dictyostelium was used as a host system to study the phenotypic and biochemical consequences associated with the mutations. The ELC carrying loop mutations bound with normal stoichiometry to the myosin heavy chain when expressed in ELC-minus cells. When expressed in wild-type cells these mutants efficiently competed with the endogenous ELC for binding, suggesting that the affinity of their interaction with the heavy chain is comparable to wild-type. However, despite apparently normal association of ELC the cells still exhibited a reduced efficiency to undergo cytokinesis in suspension. Myosin purified from these cells exhibited four to five fold reduction in actin-activated ATPase activity and a decrease in motor function as assessed by an in vitro motility assay. These results suggest that the ELC contributes to myosin's enzymatic activity in addition to providing structural support for the *-helical neck region of myosin heavy chain. --------------------------------------------------------------------- Relevance of Histone H1 Kinase Activity to the G2/M Transition during the Cell Cycle of Dictyostelium discoideum Tohru Arakane and Yasuo Maeda* Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai, 980-77 Japan J. Plant Res., in press. The implication of histone H1 kinase activity for the G2/M transition during the cell cycle was investigated using Dictyostelium discoideum Ax-2. Histone H1 kinase with its activity was purified from cell extracts by the use of p13suc1 affinity gel. In the vegetative cell cycle, the activity of histone H1 kinase including Cdc2 kinase was found using synchronized Ax-2 cells to be highest just before the entry into mitosis. The activity also was markedly enhanced just prior to the M phase from which developing cells (possibly prespore cells ) reinitiate their cell cycle at the mound-tipped aggregate stage. These results strongly suggest the importance of Cdc2 kinase activity in the G2 to M phase transition during the cell cycle, as the case for other eukaryotic cells. --------------------------------------------------------------------- Group I introns in the cytochrome oxidase genes of Dictyostelium discoideum: two related ORFs in one loop of a group I intron, cox1/2 hybrid gene and increased-size cox3 gene Shinji Ogawa, Kuniko Matsuo, Kiyohiko Angata, Kaichiro Yanagisawa and Yoshimasa Tanaka* Institute of Biological Sciences and Center for TARA*, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Curr. Genet., in press The DNA sequences of cytochrome oxidase (subunits 1, 2 and 3) genes of the cellular slime mold Dictyostelium discoideum mitochondria were determined. The genes for subunits 1 and 2 have a single continuous ORF (COX1/2) which contains four group-I introns. The insertion sites of the two group I introns (DdOX1/2.2 and DdOX1/2.3) coincide with those of fungal and algal as well as a liverwort group-I intron, in the cytochrome oxidase subunit 1. Interestingly, intron DdOX1/2.2 has two free-standing ORFs in a loop (L8) which have similar amino-acid sequences and are homologous to ai4 DNA endonuclease (I-Sce II) and bi4 RNA maturase found in group-I introns of Saccharomyces cerevisiae mitochondrial DNA. Two group-I introns (DdOX1/2.3 and DdOX1/2.4) also have a free-standing ORF in loop 1 and loop 2, respectively. These results show that these group-I introns and the intronic ORFs have evolved from the same ancestral origin, but that these ORFs have been propagated independently. --------------------------------------------------------------------- The Dictyostelium MAP Kinase ERK2 is Regulated by Ras and cAMP-Dependent Protein Kinase (PKA) and Mediates PKA Function Laurence Aubry*, Mineko Maeda*, Robert Insall, Peter N. Devreotes, and Richard A. Firtel *These two authors contributed equally to this manuscript. J. Biol. Chem., in press The chemoattractant cAMP, acting through serpentine cAMP receptors, results in a rapid and transient stimulation of the Dictyostelium MAP kinase ERK2 activity (1) . In this study we show that other pathways required for aggregation, including Ras and cAMP-dependent protein kinase (PKA), are important regulators of ERK2 activation and adaptation. By examining both the level and kinetics of activation and adaptation of ERK2, we show that Ras is a negative regulator of ERK2. Cells expressing an activated Ras protein or mutant in a Ras GAP demonstrate a reduced ERK2 activation profile while cells mutant in a putative RasGEF or expressing dominant negative Ras proteins have a more rapid, higher, and extended activation profile. We show that CRAC, a PH domain-containing protein required for the activation of adenylyl cyclase, is also required for proper adaptation of ERK2. Overexpression of PKA results in a more rapid and higher level of activation, while cells in which the PKA catalytic subunit has been disrupted show a slightly lower maximal level of activation, and more extended kinetics. Furthermore, we show that constitutive expression of PKA catalytic subunit bypasses the requirement of ERK2 for aggregation and later development. This indicates that PKA lies downstream from ERK2 and suggests that ERK2 may regulate one or more component of the signaling pathway required for mediating PKA function, possibly by directly regulating PKA R or C subunits or another protein controlling the intracellular level of cAMP. These data provide new insights into pathways by which classic G protein-coupled chemoattractant receptors regulate MAP kinase cascades and the regulatory loops controlling aggregation in Dictyostelium. --------------------------------------------------------------------- A Ras GAP is essential for cytokinesis and spatial patterning in Dictyostelium Susan Lee, Ricardo Escalante, and Richard A. Firtel DEVELOPMENT, in press Using the yeast two-hybrid system, we have identified developmentally regulated Dictyostelium genes whose encoded proteins interact with Ras.GTP but not Ras.GDP. By sequence homology and biochemical function, one of these genes encodes a Ras GAP (DdRasGAP1). Cells carrying a DdRasGAP1 gene disruption (ddrasgap1 null cells) have multiple, very distinct growth and developmental defects as elucidated by examining the phenotypes of ddrasgap1 null strains. First, vegetative ddrasgap1 null cells are very large and highly multinucleate cells when grown in suspension, indicating a severe defect in cytokinesis. When suspension-grown cells are plated in growth medium on plastic where they attach and can move, the cells rapidly become mono- and dinucleate by traction-mediated cell fission and continue to grow vegetatively with a number of nuclei (1-2) per cell, similar to that of wild-type cells. The multinucleate phenotype, combined with results indicating that constitutive expression of activated Ras does not yield highly multinucleate cells and data on Ras null mutants, suggest that Ras may need to cycle between GTP- and GDP-bound states for proper cytokinesis. After starvation, the large null cells undergo rapid fission when they start to move at the onset of aggregation, producing mononucleate cells that form a normal aggregate. Second, ddrasgap1 null cells also have multiple developmental phenotypes that indicate an essential role of DdRasGAP1 in controlling cell patterning. Multicellular development is normal through the mid-slug stage, after which morphological differentiation is very abnormal and no culminant is formed: no stalk cells and very few spores are detected. LacZ reporter studies show that by the mid-finger stage, much of the normal cell-type patterning is lost, indicating that proper DdRasGAP1 function and possibly normal Ras activity are necessary to maintain spatial organization and for induction of prestalk -> stalk and prespore -> spore cell differentiation. The inability of ddrasgap1 null cells to initiate terminal differentiation and form stalk cells is consistent with a model in which Ras functions as a mediator of inhibitory signals in cell-type differentiation at this stage. Third, DdRasGAP1 and cAMP dependent protein kinase (PKA) interact to control spatial organization within the organism. Overexpression of the PKA catalytic subunit in ddrasgap1 cells yields terminal structures that are multiply branched but lack spores. This suggests that RasGAP and PKA may mediate common pathways that regulate apical tip differentiation and organizer function, which in turn control spatial organization during multicellular development. It also suggests that DdRasGAP1 either lies downstream from PKA in the prespore -> spore pathway or in a parallel pathway that is also essential for spore differentiation. Our results indicate that DdRasGAP1 plays an essential role in controlling multiple, potentially novel pathways regulating growth and differentiation in Dictyostelium and suggest a role for Ras in these processes. --------------------------------------------------------------------- [End CSM-News, volume 7, number 15]