dictyNews Electronic Edition Volume 27, number 9 September 15, 2006 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. ============= Abstracts ============= Role of RacC for the regulation of WASP and PI3 kinase during chemotaxis of Dictyostelium Ji W. Han1, Laura Leeper1, Francisco Rivero2, Chang Y. Chung1* 1Department of Pharmacology, Vanderbilt University Medical Center, Nashville TN 37232-6600 2Zentrum fźr Biochemie and Zentrum fźr Molekulare Medizin, Medizinische FakultŠt, UniversitŠt zu Kšln, Joseph-Stlzmann-Str. 52, 50931 Kšln, Germany Journal of Biological Chemistry, in press WASP family proteins are key players for connecting multiple signaling pathways to F-actin polymerization. To dissect the highly integrated signaling pathways controlling WASP activity, we identified a Rac protein that binds to the GTPase-binding domain of WASP. Using two-hybrid and FRET-based functional assays, we identified RacC as a major regulator of WASP. RacC stimulates F-actin assembly in cell-free systems in a WASP-dependent manner. A FRET-based microscopy approach showed local activation of RacC at the leading edge of chemotaxing cells. Cells overexpressing RacC exhibit a significant increase in the level of F-actin polymerization upon cAMP stimulation, which can be blocked by a PI3 kinase inhibitor. Membrane translocation of PI3-kinase and PI(3,4,5)P3 reporter is absent in racC null cells. Cells overexpressing dominant negative RacC mutants and racC null cells move at a significantly slower speed and show a poor directionality during chemotaxis. Our results suggest that RacC plays an important role in PI3 kinase activation and WASP activation for dynamic regulation of F-actin assembly during Dictyostelium chemotaxis. Submitted by: Chang Chung [chang.chung@vanderbilt.edu] ----------------------------------------------------------------------------- Nonadaptive Regulation of ERK2 in Dictyostelium: Implications for Mechanisms of cAMP Relay. Brzostowski JA and Kimmel AR. Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-8028; Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852. Mol Biol Cell. 2006 Jul 26; (Epub ahead of print) It is assumed that ERK2 in Dictyostelium is subject to adaptive regulation in response to constant extracellular ligand stimulation. We now show, to the contrary, that ERK2 remains active under continuous stimulation, differing from most ligand-activated pathways in chemotactically-competent Dictyostelium and other cells. We show that the upstream phosphorylation pathway, responsible for ERK2 activation, transiently responds to receptor stimulation, whereas ERK2 de-phosphorylation (deactivation) is inhibited by continuous stimulation. We argue that the net result of these two regulatory actions is a persistently active ERK2 pathway when the extracellular ligand (i.e. cAMP) concentration is held constant, and that oscillatory production/destruction of secreted cAMP in chemotaxing cells accounts for the observed oscillatory activity of ERK2. We also show that pathways controlling seven-transmembrane receptor (7-TMR) ERK2 activation/deactivation function independently of G proteins and ligand-induced production of intracellular cAMP and the consequent activation of PKA. Finally, we propose that this regulation enables ERK2 to function both in an oscillatory manner, critical for chemotaxis, and in a persistent manner, necessary for gene expression, as secreted ligand concentration increases during later development. This work redefines mechanisms of ERK2 regulation by 7-TMR signaling in Dictyostelium and establishes new implications for control of signal-relay during chemotaxis. Submitted by: Joe Brzostowski [jb363a@nih.gov] ============================================================================== [End dictyNews, volume 27, number 9]