CSM News Electronic Edition Volume 7, number 3 August 3, 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 =========== Isolation and Characterization of Glycogen Synthase in Dictyostelium discoideum Brian D. Williamson, Reyna Favis, Debra A. Brickey and Charles L. Rutherford Dept. of Biology, 2119 Derring Hall, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406 Develop. Biol., in press Abstract We have partially purified the protein and isolated the glcS gene for glycogen synthase in Dictyostelium. RT-PCR results show that glcS message is present throughout the life cycle, as evidenced by the presence of a 396 bp reverse transcriptase-dependent band in both vegetative cells and during development. The protein is the product of a single gene coding for 775 amino acids, with a predicted molecular mass of 87 kDa. The presence of the three introns was confirmed by RT-PCR. The sequence is highly similar to glycogen synthase from human muscle, yeast and rat liver, diverging significantly only at the amino and carboxy termini. Phosphorylation and UDPG binding sites are conserved, with Km values for UDPG being comparable to those determined for other organisms, but in vitro phosphorylation failing to convert between the G6P-dependent (D) and -independent (I) forms. Enzyme activity is relatively constant throughout the life cycle: the I form of the enzyme isolates with the soluble fraction in amoebae, switches to the D form and becomes pellet-associated during early development, and finally reverts during late development to the I form, which again localizes to the soluble fraction. In order to study the promoter activity of the glcS gene, the 5' non-coding region of glycogen synthase and various promoter deletions were fused in frame upstream of a luciferase reporter gene. The full length glycogen synthase promoter/luc fusion extends from 1314 bp upstream to 28 bp downstream of the ATG. In agreement with the RT-PCR results, the full length promoter luciferase construct showed constant activity during development. Deletion of 485 bp (D1) and 654 bp (D2) from the 5' end of the gene resulted in a reduction to 89% and 60% of the full length promoter luciferase activity, respectively. An additional deletion of 67 bp, (deletion D3), abolishes luciferase activity. Further deletions, failed to restore luciferase activity. These results suggest that a putative regulatory element lies between deletion D2 and deletion D3. Within this 67 bp fragment there is a G-C box (CCCAACCAACCAGTG) that is similar to G-C rich sequences that have been implicated in the regulation of other Dictyostelium promoters --------------------------------------------------------------------- Activation of Dictyostelium myosin light chain kinase A by phosphorylation of threonine-166. Janet L. Smith1, Linda A. Silveira2, and James A. Spudich1 1Department of Biochemistry, Beckman Center, Stanford University Medical Center, Stanford, CA 94305-5307; 2Department of Biology, 1200 E. Colton Ave., P.O. Box 3080, University of Redlands, Redlands, CA 92373-0999 EMBO J., in press Summary Phosphorylation of the regulatory light chain is an important mechanism for the activation of myosin in nonmuscle cells. Unlike most myosin light chain kinases (MLCKs), MLCK-A from Dictyostelium is not activated by Ca2+/calmodulin. Autophosphorylation increases activity, but only to a low level, suggesting that there is an additional activation mechanism. Here we show that MLCK-A is autophosphorylated on threonine 289, which is C-terminal to the catalytic domain. Phosphorylation of MLCK-A increases in response to concanavalin A (conA) treatment of cells, which was previously shown to activate MLCK-A. However, a mutant kinase with an alanine at position 289 (T289A) is also phosphorylated in vivo, indicating that there is an additional phosphorylated residue. Based on comparisons with other protein kinases, we tested whether phosphorylation of threonine 166 drives activation of MLCK-A. Our data indicate that phosphorylation of T289 occurs in vivo, but is not associated with conA-induced activation, whereas phosphorylation of T166 by some yet unidentified kinase is associated with activation. Replacement of T166 with glutamate results in a 12-fold increase in activity as compared to the wild-type enzyme, supporting the idea that phosphorylation of T166 increases MLCK-A activity. --------------------------------------------------------------------- The Hybrid Histidine Kinase DokA is Part of the Osmotic Response System of Dictyostelium Stephan C. Schuster, Angelika A. Noegel, Felix Oehme, G=FCnther Gerisch and Melvin I. Simon EMBO J. (in press) We have used PCR to identify a Dictyostelium homolog of the bacterial two-component system. The gene dokA codes for a member of the hybrid histidine kinase family which is defined by the presence of conserved amino acid sequence motifs corresponding to an N-terminal receptor domain, a central kinase and a C-terminal response regulator moiety. Potential function of the regulator domain was demonstrated by phosphorylation in vitro. dokA mutants are deficient in the osmoregulatory pathway, resulting in premature cell death under high osmotic stress. Under less stringent osmotic conditions cells grow at normal rate, but development at the multicellular stage is altered. dokA is a member of a family of histidine kinase-like genes that play regulatory roles in eukaryotic cell function. --------------------------------------------------------------------- [End CSM News, volume 7, number 3]