dictyNews Electronic Edition Volume 34, number 6 February 19, 2010 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= Galpha5 subunit-mediated signaling requires a D-motif and the MAP kinase ERK1 in Dictyostelium Brent Raisley1, Hoai-Nghia Nguyen, and Jeffrey A. Hadwiger* Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma 74078-3020, USA Microbiology, in press The Dictyostelium Galpha5 subunit has been shown to reduce cell viability, inhibit folate chemotaxis and to accelerate tip morphogenesis and gene expression during multicellular development. Alteration of the D-motif [mitogen-activated protein kinase (MAPK) docking site] at the amino terminus of the Galpha5 subunit or the loss of the MAPK ERK1 diminished the lethality associated with the over-expression or constitutive activation of the Galpha5 subunit. The amino terminal D-motif of the Galpha5 subunit was also found necessary for the reduced cell size, small aggregate formation and precocious developmental gene expression associated with Galpha5 subunit over-expression. This D-motif also contributed to the aggregation delay in cells expressing a constitutively-active Galpha5 subunit but the D-motif was not necessary for the inhibition of folate chemotaxis. These results suggest that the amino terminal D-motif is required for some but not all phenotypes associated with elevated Galpha5 subunit functions during growth and development and that ERK1 can function in Galpha5 subunit-mediated signal transduction. Submitted by Jeff Hadwiger [jeff.hadwiger@okstate.edu] -------------------------------------------------------------------------------- MAP Kinases have different functions in Dictyostelium G Protein-Mediated Signaling Hoai-Nghia Nguyen, Brent Raisley1, and Jeffrey A. Hadwiger* Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma 74078-3020, USA Cellular Signalling, in press Extracellular signal regulated kinases (ERKs) are a class of MAP kinases that function in many signaling pathways in eukaryotic cells and in some cases, a single stimulus can activate more than one ERK suggesting functional redundancy or divergence from a common pathway. Dictyostelium discoideum encodes only two MAP kinases, ERK1 and ERK2, that both function during the developmental life cycle. To determine if ERK1 and ERK2 have overlapping functions, chemotactic and developmental phenotypes of erk1- and erk2- mutants were assessed with respect to G protein-mediated signal transduction pathways. ERK1 was specifically required for Gα5-mediated tip morphogenesis and inhibition of folate chemotaxis but not for cAMP-stimulated chemotaxis or cGMP accumulation. ERK2 was the primary MAPK phosphorylated in response to folate or cAMP stimulation. Cell growth was not altered in erk1-, erk2- or erk1-erk2- mutants but each mutant displayed a different pattern of cell sorting in chimeric aggregates. The distribution of GFP-ERK1 or GFP-ERK2 fusion proteins in the cytoplasm and nucleus was not grossly altered in cells stimulated with cAMP or folate. These results suggest ERK1 and ERK2 have different roles in G protein-mediated signaling during growth and development. Submitted by Jeff Hadwiger [jeff.hadwiger@okstate.edu] -------------------------------------------------------------------------------- Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding Loop and the Nucleotide-binding Cleft Xiong Liu, Shi Shu, Myoung-Soon S. Hong, Bin Yu and, Edward D. Korn From the Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892 J. Biol. Chem. , in press All but 11 of the 323 known actin sequences have Tyr at position 53, and the 11 exceptions have the conservative substitution Phe, which raises the questions: What is(are) the critical role(s) of Tyr-53, and, if it can be replaced by Phe, why has this happened so infrequently? We compared the properties of purified endogenous Dictyostelium actin and mutant constructs with Tyr-53 replaced by Phe, Ala, Glu, Trp and Leu. The Tyr53Phe mutant did not differ significantly from endogenous actin in any of the properties assayed, but the Tyr53Ala and Tyr53Glu mutants differed substantially: affinity for DNase I was reduced, the rate of nucleotide exchange was increased, the critical concentration for polymerization was increased, filament elongation was inhibited, and polymerized actin was in the form of small oligomers and imperfect filaments. Growth and/or development of cells expressing these actin mutants were also inhibited. The Trp and Leu mutations had less, but still significant, effects. We conclude that either Tyr or Phe is required to maintain the functional conformations of the DNase I-binding loop (D-loop), and that the conformation of the D-loop affects not only the properties that directly involve the D-loop but also allosterically modifies the conformation of the nucleotide-binding cleft. The apparent evolutionary “preference” for Tyr at position 53 may be because Tyr allows dynamic modification of the D-loop conformation by phosphorylation (Baek et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 11748-11753) with effects similar, but not identical, to those of the Ala and Glu mutations. Submitted by Edward Korn [korned2@nhlbi.nih.gov] -------------------------------------------------------------------------------- Identification of a new mechanism for targeting myosin II heavy chain phosphorylation by Dictyostelium myosin heavy chain kinase B Julie Underwood, Jonathan Greene, and Paul A. Steimle Department of Biology, University of North Carolina at Greensboro, Greensboro, North Carolina, USA BMC Research Notes, in press BACKGROUND: Heavy chain phosphorylation plays a central role in regulating myosin II bipolar filament assembly in Dictyostelium, as well as in higher eukaryotic nonmuscle cells. Our previous work has demonstrated that the WD-repeat domain of Dictyostelium myosin II heavy chain kinase B (MHCK-B), unlike its counterpart in MHCK-A, is not absolutely required for targeting of the kinase to Phosphorylate MHC. Thus, we tested the hypothesis that an asparagine-rich and structurally disordered region that is unique to MHCK-B can by itself function in substrate targeting. FINDINGS: Biochemical assays comparing the activities of full-length MHCK-B, a truncation lacking only the WD-repeat domain (B-delta-WD), and a truncation lacking both the N-rich region and the WD-repeat domain (B-delta-N-WD) revealed that the N-rich region targets MHCK-B to phosphorylate MHC in a manner that leads to bipolar filament disassembly. This targeting is physiologically relevant since cellular over-expression of the B-delta-WD truncation, but not the B-delta-N-WD truncation, leads to dramatically reduced levels of myosin II filament assembly and associated defects in cytokinesis and multicellular development. CONCLUSIONS: The results presented here demonstrate that an intrinsically unstructured, and asparagine-rich, region of a MHCK-B can mediate specific targeting of the kinase to phosphorylate myosin II heavy chain. This targeting involves a direct binding interaction with myosin II filaments. In terms of regulating myosin bipolar filament assembly, our results suggest that factors affecting the activity of this unique region of MHCK-B could allow for regulation of MHCKB in a manner that is distinct from the other MHCKs in Dictyostelium. Submitted by Paul Steimle [p_steiml@uncg.edu] ============================================================== [End dictyNews, volume 34, number 6]