CSM News Electronic Edition Volume 8, number 10 April 26, 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by World Wide Web at the URL "http://dicty.cmb.nwu.edu/dicty/dicty.html" Abstracts H+ secretion induced by hypertonic stress in the cellular slime mold Dictyostelium discoideum Masakazu Oyama* and Kou Kubota Department of Life Science, Faculty of Science, Himeji Institute of Technology, Shosha 2167, Himeji 671-22 Japan J. Biochem. in press We found that Dictyostelium amebas shrank about 50% in 10 min after a change in extracellular osmolarity from 70 mosM to 170-370 mosM. Cells shrunk by the addition of 0.1 M mannitol showed a partial recovery of cell volume in 2 h when the culture medium contained salts. A full recovery of cell volume was observed when 0.1 or 0.2 osM NaCl or KCl was added instead of mannitol. No recovery of cell volume was observed after the addition of 0.1 M mannitol or glycine when the extracellular medium did not contain any salt. An elevation in extracellular pH of starved Dictyostelium cells, which is probably due to ammonia production, was suppressed by hypertonic stress. This hypertonic stress-induced suppression of the elevation in extracellular pH was observed only when the extracellular medium contained salt. We also found that cells with artificially elevated intracellular pH withstood hypertonic stress much better than control cells. These results suggest that Dictyostelium cells under hypertonic conditions secrete protons and take up charged extracellular osmolytes in exchange. The uptake of charged osmolytes induces the elevation of intracellular osmotic strength and the recovery of cell volume. The elevation of intracellular pH, which is presumably induced by the proton secretion, plays a role in protecting cells from lysis. Phosphorylation of the G protein a-subunit, Ga2, of Dictyostelium discoideum requires a functional and activated Ga2. Robert E. Gundersen Department of Biochemistry, Microbiolgy, and Molecular Biology University of Maine, Orono, Maine 04469-5735 J. Cellular Biochemistry, in press. The Ga2-subunit of Dictyostelium discoideum is essential to the initial stage of the cells' developmental life cycle. In response to the extracelluar chemoattractant, cAMP, Ga2 is activated and transiently phosphorylated on serine-113 (Chen et al., 1994). The role of Ga2 phosphorylation remains elusive; cells expressing the S113A, nonphosphorylated mutation of Ga2 appear to proceed through the developmental phase normally. To gain insight into the fucntion of Ga2 phosphorylation, the conditions for Ga2 phosphorylation were examined using a variety of a- subunit point mutations and chimeras. Mutations that block the G protein activation cycle prior to or at the hydrolysis of GTP (Ga2-S45A, Ga2-G207A and Ga2- Q208L) preclude Ga2 phosphorylation in vivo. Phosphorylation of the Ga2-Q208L mutation does however occur in an in vitro phosphorylation assay. It appears that Ga2 phosphorylation, in vivo shown previously to require the cAMP receptor, also requires signaling through the G2 pathway. Results from the in vitro assay suggest that the substrate for phosphorylation is the a-subunit monomer. Identification of a Protein Kinase from Dictyostelium with Homology to the Novel Catalytic Domain of Myosin Heavy Chain Kinase A. Colleen E. Clancy, Manual G. Mendoza, Teresa V. Naismith, Michael F. Kolman, and Thomas T. Egelhoff# Department of Physiology and Biophysics, Case Western Reserve School of Medicine, Cleveland, OH. 44106- 4970 J. Biol. Chem. , in press SUMMARY Myosin II assembly and localization into the cytoskeleton is regulated by heavy chain phosphorylation in Dictyostelium. The enzyme Myosin Heavy Chain Kinase A (MHCK A) has been shown previously to drive myosin filament disassembly in vitro and in vivo. MHCK A is noteworthy in that its catalytic domain is unrelated to the conventional families of eukaryotic protein kinases. We report here the cloning and initial biochemical characterization of another kinase from Dictyostelium which is related to MHCK A. When the segment of this protein that is similar to the MHCK A catalytic domain was expressed in bacteria, the resultant protein displayed efficient autophosphorylation, phosphorylated Dictyostelium myosin II, and also phosphorylated a peptide substrate corresponding to a portion of the myosin II tail. We have therefore named this gene myosin heavy chain kinase B. These results provide the first confirmation that sequences in other proteins which are related to the MHCK A catalytic domain can also encode protein kinase activity. It is likely that the related segments of homology present in rat eukaryotic elongation factor-2 (eEF-2) kinase and a putative nematode eEF-2 kinase also encode the catalytic domains of those enzymes. Dictyostelium Myosin Heavy Chain Kinase A Subdomains: coiled-coil and WD-repeat roles in oligomerization and substrate targeting Michael F. Kolman and Thomas T. Egelhoff* Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio, 44106-4970. J. Biol. Chem. In press Summary Myosin heavy chain kinase A participates in the regulation of cytoskeletal myosin assembly in Dictyostelium, driving filament disassembly via phosphorylation of sites in the myosin tail. MHCK A contains an amino-terminal coiled-coil domain, a novel central catalytic domain, and a carboxyl-terminal domain containing a 7-fold WD-repeat motif. We have overexpressed MHCK A truncation constructs to clarify the roles of each of these domains. Recombinant full length MHCK A, MHCK A lacking the predicted coiled-coil domain (delta-Coil- MHCK A), and MHCK A lacking the WD-repeat domain (delta-WD-MHCK A) were expressed at high level in Dictyostelium cells lacking endogenous MHCK A. Cellular analysis, together with biochemical analysis of the purified proteins demonstrates that the putative coiled- coil domain is responsible for the oligomerization of the MHCK A holoenzyme. Removal of the WD-repeat domain had no effect on catalytic activity towards a synthetic peptide, but did result in a 95% loss of protein kinase activity when native myosin filaments were used as the substrate. Cellular analysis confirms that the same severe loss of activity against myosin occurs in vivo when the WD-repeat domain is eliminated. These results suggest that the WD-repeat domain of MHCK A serves to target this enzyme to its physiological substrate. [End CSM News, volume 8, number 10]