CSM News Electronic Edition Volume 8, number 3 January 25, 1997 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 =========== Myosin I Overexpression Impairs Cell Migration. Kristine D. Novak & Margaret A. Titus Department of Cell Biology, Duke University Medical Center, Durham, NC 27710 J. Cell Biology, in press Abstract Dictyostelium myoB, a member of the myosin I family of motor proteins, is important for controlling the formation and retraction of membrane projections by the cell's actin cortex (Novak et al., J. Cell Biol. 131:1205). Mutants that express a 3 - 7 fold excess of myoB (myoB+ cells) were generated to further analyze the role of myosin I in these processes. The myoB+ cells move with an instantaneous velocity that is 35% of the wild-type rate and exhibit a 6 - 8 hour delay in initiation of aggregation when placed under starvation conditions. The myoB+ cells complete the developmental cycle after an extended period of time, but they form fewer fruiting bodies that appear to be small and abnormal. The myoB+ cells are also deficient in their ability to form distinct F-actin filled projections such as crowns, or become elongate and polarized. This defect can be attributed to the presence of at least 3 - fold more myoB at the cortex of the myoB+ cells. In contrast, three-fold overexpression of a truncated myoB that lacks the SH3 domain (myoB/SH3- cells) or myoB in which the consensus heavy chain phosphorylation site was mutated to an alanine ( S332A-myoB) does not disturb normal cellular function. However, there is an increased concentration of myoB in the cortex of the myoB/SH3- and S332A-myoB cells comparable to that found in the myoB+ cells. These results suggest that excess full-length cortical myoB prevents the formation of the actin-filled extensions required for locomotion by increasing the tension of the F-actin cytoskeleton and/or retracting projections before they can fully extend. They also demonstrate a role for the phosphorylation site and SH3 domain in mediating the in vivo activity of myosin I. --------------------------------------------------------------------- Mapping of the Novel Protein Kinase Catalytic Domain of Dictyostelium Myosin II Heavy Chain Kinase A. Graham P. Cote1, Xia Luo1, Michael B. Murphy2, and Thomas T. Egelhoff2* 1) Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6 and 2) Department of Physiology and Biophysics, Case Western Reserve University Cleveland, Ohio 44106 J. Biol. Chem., in press. ABSTRACT Myosin heavy chain kinase A (MHCK A) in Dictyostelium was identified as a biochemical activity that phosphorylates threonine residues in the myosin II tail domain and regulates myosin filament assembly. The catalytic domain of MHCK A has now been mapped through the functional characterization of a series of MHCK A truncation mutants expressed E. coli. A recombinant protein comprising the central non-repetitive domain of MHCK A (residues 552-841) was isolated in a soluble form and shown to phosphorylate Dictyostelium myosin II, myelin basic protein and a synthetic peptide substrate. The functionally mapped catalytic domain of MHCK A shows no detectable sequence similarity to known classes of eukaryotic protein kinases but shares substantial sequence similarity with a transcribed C. elegans gene and with the mammalian elongation factor-2 kinase (calcium/calmodulin-dependent protein kinase III). We suggest that MHCK A represents the prototype for a novel, widely occurring protein kinase family. --------------------------------------------------------------------- Cloning and characterization of the gene encoding a mitochondrially localized DNA topoisomerase II in Dictyostelium discoideum : Western blot analysis Kayoko Komori a, Kenji Kuroe a, Kaichiro Yanagisawa a and Yoshimasa Tanaka a, b a Institute of Biological Sciences and b Center for TARA, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Biochim. Biophys. Acta, in press Abstract We cloned a gene (topA) encoding DNA topoisomerase II from Dictyostelium discoideum nuclear DNA using oligo probes corresponding to the consensus amino acid sequences found in the gene in other eukaryotes. The gene encoding a predicted polypeptide of 1282 amino acids with Mr of about 146 kDa, is a single copy that is expressed as a polyadenylated 4.5 kb RNA. The predicted amino acid sequence shares similarity with those of other eukaryotes with identity between 32 and 46%. The protein is 260-300 amino acids shorter in the C-terminal region and 50-80 longer in the N-terminal region than those of other eukaryotes. In TopA of D. discoideum, the N-terminal region with stretches of charged and hydrophilic amino acids is predicted to fold into an amphiphilic a-helix which is characteristic of a mitochondrial targeting signal presequence. Four independent polyclonal antibodies against bacterially expressed GST fusion proteins containing four portions of the polypeptide detected a single band on Western blots at about 135 kDa. Western blots analysis of subcellular fractions revealed that this protein is localized in mitochondria. The protein and the mRNA are present in growth phase and during development, although levels of both declined as development proceeded. ------------------------------------------------------------------------------- [End CSM-News, volume 8, number 3]