Dicty News Electronic Edition Volume 11, number 6 Sept. 26, 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" ========================= Pictures from Dicty98 ========================= We are looking for contributions to the photo archive for the Dicty web page. If you have any photos you'd like to contribute you can send them electronically in tif, jpg or gif formats, as a photoshop document or if you prefer you can mail copies and we will scan them. The photos of previous meetings are one of the most frequently viewed parts of the web site, so it would be great to have a good collection of pictures from Dicty98. Send files to "dicty@nwu.edu". ================ Announcement ================ A report by Bill Loomis on the Dictyostelium genome sequencing project has been added to the Dictyostelium home page. The link is: http://dicty.cmb.nwu.edu/dicty/Genomeseq.htm =========== Abstracts =========== Regulation of the PAK-related Dictyostelium Myosin I Heavy Chain Kinase by Autophosphorylation, Acidic Phospholipids and Ca2+-Calmodulin* Sheu-Fen Lee, Amjad Mahasneh, Marc de la Roche and Graham P. C�t� Department of Biochemistry, Queen�s University, Kingston, Ontario, Canada, K7L 3N6 J. Biol. Chem., In Press Abstract The Dictyostelium myosin I heavy chain kinase (MIHCK) is a member of the p21-activated (PAK) kinase family (Lee, S-.F., et al. (1996) J.Biol.Chem. 271, 27044). MIHCK incubated with MgATP in the absence of effectors incorporates 1 mol of phosphate per mol resulting in an ~40-fold increase in kinase activity. Sequence analysis of tryptic peptides has identified the major site of phosphorylation as Ser-8. A peptide and a GST-fusion protein containing the Ser-8 phosphorylation site were good substrates for MIHCK, indicating that MIHCK can catalyze its own activation. GTP S-Rac1 stimulates MIHCK autophosphorylation and kinase activity 10-fold. Phosphatidylserine, phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate, but not phosphatidylcholine or sphingosine, were as effective as GTP S-Rac1 in enhancing MIHCK autophosphorylation and activity. Acidic lipids and GTP S-Rac1 induced the autophosphorylation of a similar set of sites as judged by two-dimensional tryptic peptide maps. It is proposed that GTP-Rac and acidic phospholipids function cooperatively to associate MIHCK with membranes. Ca2+-calmodulin bound MIHCK and inhibited activation by acidic phospholipids but not GTP S-Rac1. These studies reveal a number of similarities between the regulatory properties of the Dictyostelium and Acanthamoeba MIHCK, suggesting that the signaling pathways that control myosin I are conserved. ------------------------------------------------------------------------- Myosin light chain kinase A, an unconventional myosin light chain kinase from Dictyostelium, is activated by a cGMP-dependent pathway Linda A. Silveira*#, Janet L. Smith*, John L. Tan*, and James A. Spudich* *Department of Biochemistry, Beckman Center, Stanford University, Stanford CA 94305-5307, #Department of Biology, University of Redlands, 1200 E. Colton Avenue, P.O. Box 3080, Redlands CA 92373-0999 Proc. Natl. Acad. Sci., in press Abstract Dictyostelium myosin II is activated by phosphorylation of its regulatory light chain by MLCK-A, an unconventional myosin light chain kinase that is not regulated by Ca2+/calmodulin. MLCK-A is activated by autophosphorylation of threonine-289 outside of the catalytic domain and by phosphorylation of threonine-166 in the activation loop by an unidentified kinase, but the signals controlling these phosphorylations are unknown. Treatment of cells with concanavalin A results in quantitative phosphorylation of the regulatory light chain by MLCK-A, providing an opportunity to study MLCK-A�s activation mechanism. MLCK-A does not alter its cellular location upon treatment of cells with concanavalin A, nor does it localize to the myosin-rich caps that form after treatment. However, MLCK-A activity rapidly increases two to 13-fold when Dictyostelium cells are exposed to concanavalin A. This activation can occur in the absence of MLCK-A autophosphorylation. cGMP is a promising candidate for an intracellular messenger mediating conA-triggered MLCK-A activation, as addition of cGMP to fresh Dictyostelium lysates increases MLCK-A activity three to 12-fold. The specific activity of MLCK-A in cGMP-treated lysates is 210-fold higher than that of recombinant MLCK-A, which is fully autophosphorylated, but lacks threonine-166 phosphorylation. Purified MLCK-A is not directly activated by cGMP, indicating that additional cellular factors, perhaps a kinase that phosphorylates threonine-166, are involved. ------------------------------------------------------------------------- Induction of optical density waves and chemotactic cell movement in Dictyostelium discoideum by micro-injection of cAMP pulses Jens Rietdorf, Florian Siegert and Cornelis J. Weijer Dept. of Anatomy & Physiology, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, UK Develop. Biol., in press SUMMARY The development of most multicellular organisms involves coordinated cell movement. The early aggregation of Dictyostelium cells has been shown to be mediated by chemotactic movement to propagating waves of cAMP. We have proposed that propagating waves of a chemoattractant, most likely cAMP, also controls the movement of cells in mounds and slugs (Bretschneider et al., 1995; Siegert and Weijer, 1995). We have now used periodic pressure injection of pulses of cAMP in the extracellular space of aggregation streams, mounds and slugs to investigate whether these signals can be relayed and control cell movement, using quantitative digital time-lapse microscopy. Our major findings are (1) Short (0.1 sec) pulses of cAMP (107 molecules) were able to elicit optical density (OD) waves in fields of aggregating amoebae. They propagate from the micropipette outward and interact with endogenous OD waves. (2) Periodic injection of cAMP pulses into aggregation streams blocked the pulses coming from the center and led to the rapid accumulation of cells downstream of the pipette around the pipette. (3) Injection of pulses of cAMP into mounds elicited OD waves, which propagated from the pipette outward and interacted with the endogenous waves, indicating that the same propagator carries them. (4) Periodic microinjection of cAMP in the prespore zone of slugs led to accumulation of anterior-like cells around the micropipette followed by tip formation. Furthermore, the cAMP signal could control the spacing of the endogenous sorting pattern. These results strongly support the hypothesis that the optical density waves observed during early development up to the mound stage represent propagating cAMP waves. They suggest furthermore that cAMP is the morphogen that controls cell movements in slugs. ------------------------------------------------------------------------- The Characterization of Two Dictyostelium discoideum Genes Encoding Ribosomal Proteins with Sequence Similarity to Rat L27a and L37a Tetsuo OHMACHI 1*, Ryo FUKUOKA 1, Yoshie KIMURA 1, Yoshihiro ASADA 1 and Herbert L. ENNIS 2 1 Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan 2 Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA Biosci. Biotechnol. Biochem. in press Summary Two Dictyostelium discoideum ribosomal protein genes, denoted DdL27a and DdL37a, were isolated and sequenced. The DdL27a gene contained an open reading frame of 148 amino acids coding for a putative 16,407 Da protein, which was similar to rat L27a (82.6% similarity) and to ribosomal proteins from other species. The gene contained a 311-bp intron downstream from the ATG initiation codon with an A+T content of 75%. The DdL37a gene encoded a 9,999 Da protein consisting of 91 amino acids, which had high sequence similarity to rat, human, and chicken ribosomal protein L37a, and was interrupted by two introns of 254 bp and 75 bp in length. The DdL37a protein contained a typical zinc finger motif (Cys-X2-Cys-X14- Cys-X2-Cys), which may be involved in the interaction of proteins with nucleic acids. Genomic DNA blot analysis indicated that the DdL27a and DdL37a genes are present in single copies in the Dictyostelium haploid genome. The DdL27a and DdL37a mRNA were expressed maximally in growing amoebae, and their levels decreased during multicellular development, coordinately with the observed decrease in ribosome accumulation during later development. ------------------------------------------------------------------------- Cloning, sequencing and developmental expression of the genes encoding S4 and S10 ribosomal proteins in the cellular slime mould Dictyostelium discoideum Aurelie Tapparo, Michel Satre and Gerard Klein Laboratoire de Biochimie et de Biophysique des Systemes Integres (UMR 314 CNRS-CEA), Departement de Biologie Moleculaire et Structurale, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 09, France Current Genetics, in press Abstract The sequences of the rps4 and rps10 genes encoding the Dictyostelium discoideum homologues of the basic ribosomal proteins S4 and S10 were determined from cDNA and genomic DNA clones. They are expressed respectively as 266 and 153 amino acid-long proteins. In both cases, the N-terminal methionine is cleaved in the mature proteins. S4 contains two putative nuclear targeting signals and displays a strong overall identity (around 60%) to eukaryotic S4 homologues. The rps10 gene harbours a 314 bp intron located close to its 5�-coding end. The overall identity between D. discoideum S10 and eukaryotic homologues is around 38% and rises to 53% in the N-terminal domain. Southern blots suggest that both S4 and S10 are encoded by single genes that are regulated during development. The corresponding mRNAs decrease sharply after 8 h of differentiation. ------------------------------------------------------------------------- A novel, putative MEK kinase controls developmental timing and spatial patterning in Dictyostelium and is regulated by ubiquitin-mediated protein degradation Chang Y. Chung, T. B. K. Reddy, Kemin Zhou, and Richard A. Firtel Department of Biology, Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634 Genes Dev. in press. ABSTRACT We have identified a developmentally regulated, putative MEK kinase (MEKKa) that contains an F-box and WD40 repeats and plays a complex role in regulating cell-type differentiation and spatial patterning. Cells deficient in MEKKa develop precociously and exhibit abnormal cell-type patterning with an increase in one of the prestalk compartments (pstO), a concomitant reduction in the prespore domain, and a loss of the sharp compartment boundaries, resulting in overlapping prestalk and prespore domains. Overexpression of MEKKa or MEKKa lacking the WD40 repeats results in very delayed development and a severe loss of compartment boundaries. Prespore and prestalk cells are interspersed throughout the slug. Analysis of chimeric organisms suggests that MEKKa function is required for the proper induction and maintenance of prespore cell differentiation. We show that the WD40 repeats target MEKKa to the cortical region of the cell, while the F-box/WD40 repeats direct ubiquitin-mediated MEKKa degradation. We identify a UBC and a UBP (ubiquitin hydrolase) that interact with the F-box/WD40 repeats. Our findings indicate that cells lacking the ubiquitin hydrolase have phenotypes similar to those of MEKKa null (mekka-) cells, further supporting a direct genetic and biochemical interaction between MEKKa, the UBC, and the UBP. We demonstrate that UBC and UBP differentially control MEKKa ubiquitination/deubiquitination and degradation through the F-box/WD40 repeats in a cell-type-specific and temporally-regulated manner. Our results represent a novel mechanism including targeted protein degradation by which MAP kinase cascade components can be controlled. More importantly, our findings suggest a new paradigm of spatial and temporal control of the kinase activity controlling spatial patterning during multicellular development, which parallels the temporally-regulated degradation of proteins required for cell-cycle progression. ------------------------------------------------------------------------- [End Dicty News, volume 11, number 6]