Dicty News Electronic Edition Volume 14, number 13 June 24, 2000 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 ============== Isolation and characterization of casein kinase I from Dictyostelium discoideum Gema Moreno-Bueno, Carmela Calés, M. Margarita Behrens§ and Margarita Fernández-Renart* Biochem. J., in press. SUMMARY In the present study, the molecular cloning and characterization of a 49-kDa form of casein kinase I (CKI) from Dictyostelium discoideum is reported. The predicted aminoacid sequence shares 70% identity with the catalytic domain of the mammalian d and e isoforms, Drosophila CKIe and Schizosaccharomyces pombe Hhp1, and 63% identity with Hrr25, a 57-kDa form of yeast casein kinase involved in DNA repair. D. discoideum CKI (DdCKI) was expressed in vegetative asynchronous cells as well as in differentiated cells, as detected by Northern-blot analysis. The level of DdCKI expression does not change during the cell cycle. Antibodies raised against a truncated version of the protein recognize a 49-kDa protein from D. discoideum extracts. Protein expression parallels the pattern found for the RNA. The expression of D. Discoideum CKI in Escherichia coli resulted in an active enzyme that autophosphorylates and phosphorylates casein. Immunofluorescence assays show that D.discoideum CKI is localized in the cytoplasm and nuclei of Dictyostelium cells. The lack of disruptants of the CKI gene suggests that this protein is essential for the vegetative growth of D. discoideum. Overxepression of D.discoideum CKI results in cells with increased resistance to hydroxyurea, suggesting a potential role for this kinase in DNA repair. ---------------------------------------------------------------------------- Involvement of the glucose-regulated protein 94 (Dd-GRP94) in starvation response of Dictyostelium discoideum cells Tsuyoshi Morita*, Kenji Saitoh, Takashi Takagi and Yasuo Maeda Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai 980-8578, Japan Biochemical and Biophysical research communications, in press. SUMMARY Upon deprivation of nutrients, Dictyostelium discoideum Ax-2 cells arrest proliferation and initiate a metamorphosed developmental program including induction of altered gene expressions which are necessary for differentiation. In Ax-2 cells, we found out a member of Hsp90 family usually contained in the endoplasmic reticulum (ER), Dd-GRP94 (Dictyostelium discoideum glucose-regulated protein 94). In general, GRP94 are induced either by glucose-depletion or by depletion of Ca2+ in intracellular Ca2+ stores. Unexpectedly, however, the expression of Dd-grp94 was greatly reduced within 60 minutes of starvation. Dd-grp94-overexpressing cells (GRP94OE cells) collected without forming distinct aggregation-streams, and never formed normal fruiting bodies. Also, prespore differentiation as well as maturation into spores and stalk cells were particularly impaired in the GRP94OE cells. Thus Dd-GRP94 seems to be crucial in late differentiation as well as in starvation response. ---------------------------------------------------------------------------- Pyridoxal kinase knockout of Dictyostelium complemented by the human homologue Kunde Guo and Peter C. Newell Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK FEMS Microbiol. Letts., in press. SUMMARY The gene (pykA) coding for pyridoxal kinase which converts pyridoxal (vitamin B6) to pyridoxal phosphate, was isolated from Dictyostelium discoideum using insertional mutagenesis. Pyridoxal phosphate is a co-factor for the amino acid transamination reaction, which is particularly important for Dictyostelium as it depends on protein degradation for survival during development. PykA knockout cells were agg-minus on SM plates and grew poorly in axenic medium with low yield but growth was restored by the addition of pyridoxal phosphate. Sequencing indicated a gene, with one intron, coding for a predicted protein of 301 amino acids that was 42% identical in amino acid sequence to human pyridoxal kinase. After expression of the wild type gene in E. coli, the purified PykA protein product was shown to have pyridoxal kinase enzymatic activity with a Km of 8.7 mM for pyridoxal. Transformation of the Dictyostelium knockout mutant with the human pyridoxal kinase gene gave almost complete complementation of growth and development in a similar manner to that seen using transformation with the wild type Dictyostelium gene. Phylogenetic analysis indicated that the Dictyostelium amino acid sequence was closer to human pyridoxal kinase than to pyridoxal kinases of lower eukaryotes. ---------------------------------------------------------------------------- CBP1 Associates with the Dictyostelium Cytoskeleton and Is Important for Normal Cell Aggregation under Certain Developmental Conditions Akil Dharamsi, diane Tessarolo, Barrie Coukell and Jason Pun Department of Biology, York University, Toronto, Ontario, M3J 1P3, Canada Exp. Cell Res. (in press) In cells of the eukaryotic microorganism, Dictyostelium discoideum, at least eight small, four EF-hand Ca2+-binding proteins of unknown function are expressed at specific times during development. One of these proteins, calcium-binding protein 1 (CBP1), first appears just prior to cell aggregation and then is present at relatively constant levels throughout development. To determine a role for CBP1 during development, the protein was used as bait in a yeast two-hybrid screen to reveal putative CBP1-interacting proteins. Two proteins identified in this screen were the actin-binding proteins, protovillin and EF-1a . Using an in vitro binding assay, both of these proteins were found to interact with CBP1 in the absence of Ca2+, but the interaction of CBP1 with EF-1a was increased substantially by Ca2+. CBP1 was also shown by fluorescence microscopy and by binding assays to associate with the actin cytoskeleton of Dictyostelium cells during development, and these interactions were partially Ca2+ dependent. cbpA-null cells grew normally, but under certain developmental conditions, cell aggregation was prolonged and irregular. This defect in aggregation appeared to be related to a general reduction in cell motility rather than to a decrease in the ability of the cells to respond to the chemoattractant, cAMP. Together, these results suggest that CBP1 might function to help regulate the reorganization of the Dictyostelium actin cytoskeleton during cell aggregation. ---------------------------------------------------------------------------- A novel role of differentiation-inducing factor-1 (DIF-1) in Dictyostelium development, assessed by the restoration of a developmental defect of the mutant lacking MAP-kinase, ERK2 Hidekazu Kuwayama,1 Masakazu Oyama,2 Yuzuru Kubohara,3 and Mineko Maeda1* 1) Department of Biology, University of Osaka, Machikaneyama-cho 1-16, Toyonaka, Osaka 560-0043, Japan. 2) Department of Life Science, Faculty of Science, Himeji Institute of Technology, Shosha 2107, Himeji, Hyogo 671-2201, Japan. 3) Biosignal Research Center, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Showa-machi 3-39-15, Maebashi, 371-8512, Japan. Development, Growth & Differentiation (in press) Summary We have previously reported that the differentiating wild-type cells of Dictyostelium discoideum secrete a diffusible factor(s) which is able to rescue the developmental defect in the mutant lacking MAP-kinase ERK2 encoded by the gene erkB. In this study, we demonstrate that DIF-1 (differentiation- inducing factor for stalk cells) can mimic the role of the factor(s), and we also discuss the mechanism of the action of DIF-1 in the erkB-null mutant. The mutant usually never form multicellular aggregates because of its defect in cAMP signaling. In the presence of 100 nM DIF-1, however, the mutant cells formed tiny slugs, which eventually developed into small fruiting bodies. By contrast, DIF-1 never rescued the developmental arrest of other Dictyostelium mutants lacking adenylyl cyclase A (ACA), cAMP receptors (cAR1 and cAR3), heterotrimeric G protein, the cytosolic regulator of ACA (CRAC), or the catalytic subunit of cAMP-dependent protein kinase (PKA-C). Most importantly, it was found that DIF-1 did not affect the cellular cAMP level but rather elevated the transcriptional level of pka during the development of erkB-null cells. These results suggest that DIF-1 may rescue the developmental defect in erkB-null cells via the increase in PKA activity, thus giving the first conclusive evidence that DIF-1 plays a crucial role in the early event of Dictyostelium development as well as in prestalk- and stalk-cell induction. ---------------------------------------------------------------------------- [End Dicty News, volume 14, number 13]