dictyNews Electronic Edition Volume 31, number 7 August 15, 2008 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. Upon publication of your paper, please send strains and plamids to  the Dicty Stock Center. For more information see  http://dictybase.org/StockCenter/Deposit.html. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ========= Abstracts ========= Dictyostelium kinase DPYK3 negatively regulates STATc signaling in cell  fate decision. Lee NS, Rodriguez M, Kim B, Kim L. Department of Biological Sciences, Florida International University,  Miami, FL 33199 USA. Dev Growth Differ. in press DPYK3, a member of the Dictyostelium TKL (tyrosine kinase like) kinase  family, was ablated by homologous recombination. dpyk3(-) cells displayed  aberrant pattern formation during development. The prestalk O zone was  not properly formed and, instead, the prespore zone was expanded in  dpyk3(-) slugs. During development, the transcription factor STATc  (signal transducers and activators of transcription c) was persistently  phosphorylated and ecmAO expression level was kept low in dpyk3(-) cells.  Furthermore, in response to differentiation inducing factor-1 (DIF-1) in  suspension culture, dpyk3(-) cells displayed persistent STATc phosphorylation  and reintroduction of DPYK3 in dpyk3(-) cells restored transient STATc  phosphorylation similarly to wild type cells. In contrast to the positive  STAT regulation by Janus Kinase in metazoans, Dictyostelium DPYK3  negatively regulates STATc during development in response to  DIF-1 signaling. Submitted by: Marbelys Rodriguez [mrodr126@fiu.edu] -------------------------------------------------------------------------------- Cooperation between ENTH and Carboxy-terminal Domains of Dictyostelium Epsin Regulates Dynamic Interaction with Clathrin-Coated Pits Rebecca J. Brady, Yujia Wen, and Theresa J. O’Halloran Department of Molecular Cell and Developmental Biology, Institute for Cellular  and Molecular Biology, University of Texas at Austin, Austin, TX 78712 email:  t.ohalloran@mail.utexas.edu Journal of Cell Science Epsin contains a phospholipid-binding ENTH domain coupled to a  carboxy-terminal domain containing motifs that bind coated pit proteins. We  examined how these domains interact to influence Dictyostelium epsin function  and localization. While not required for global clathrin function, epsin was  essential for constructing oval spores during development. Within the epsin  protein, we found that features important for essential function were distinct from  features targeting epsin to clathrin-coated pits. On its own, the  phospholipid-binding ENTH domain could rescue the epsin null phenotype.  While necessary and sufficient for function, the isolated ENTH domain was  not targeted within clathrin coated pits. The coated pit motif-containing  C-terminal domain was also insufficient, highlighting a requirement for both  domains for targeting to coated pits. Replacement of the ENTH domain by an  alternate membrane-binding domain led to an epsin that sequestered clathrin  and AP2 and ablated clathrin function, supporting a modulatory role for the  ENTH domain. Within the ENTH domain, residues important for PIP2-binding  were essential for both epsin localization and function, while residue T107  was essential for function but not coated pit localization. Our results  support a model where the ENTH domain coordinates with the clathrin-binding  c-terminal domain to allow a dynamic interaction of epsin with coated pits. Submitted by: Terry O’Halloran [t.ohalloran@mail.utexas.edu] -------------------------------------------------------------------------------- Intramolecular activation mechanism of the Dictyostelium LRRK2-homolog  Roco protein GbpC Wouter N. van Egmond, Arjan Kortholt, Katarzyna Plak, Leonard Bosgraaf,  Sylvia Bosgraaf, Ineke Keizer-Gunnink and Peter J.M. van Haastert Department of Cell Biochemistry, University of Groningen Kerklaan 30,  9751 NN Haren, The Netherlands J. Biol. Chem, in press GbpC is a large multidomain protein involved in cGMP-mediated chemotaxis in  the cellular slime mold Dictyostelium discoideum. GbpC belongs to the Roco  family of proteins that often share a central core region, consisting of  Leucine-Rich-Repeats, a Ras domain (Roc), a COR domain and a MAPKKKinase  domain. In addition to this core, GbpC contains a RasGEF domain and two  cGMP-binding domains. Here, we report on an intramolecular signaling cascade  of GbpC. In vitro, the RasGEF domain of GbpC specifically accelerates the  GDP/GTP exchange of the Roc domain. Moreover, cGMP-binding to GbpC strongly  stimulates the binding of GbpC to GTP-agarose, suggesting cGMP-stimulated  GDP/GTP exchange at the Roc domain. The function of the protein in vivo was  investigated by rescue analysis of the chemotactic defect of gbpC null cells.  Mutants that lack a functional GEF, Roc or kinase domain are inactive in vivo.  Together, the results suggest a 4-step intramolecular activation mechanism of the  Roco protein GbpC: cGMP-binding to the cNB domains, activation of the GEF  domain, GDP/GTP exchange of Roc and activation of the MAPKKK domain.   Submitted by: Peter van Haastert [p.j.m.van.haastert@rug.nl] -------------------------------------------------------------------------------- Structure of the Roc–COR domain tandem of C. tepidum, a prokaryotic homologue  of the human LRRK2 Parkinson kinase Katja Gotthardt1, Michael Weyand1, Arjan Kortholt1, 2, Peter J M Van Haastert2  and Alfred Wittinghofer1 1 Department of Structural Biology, Max-Planck-Institut for Molecular  Physiology, Dortmund, Germany 2 Department of Cell Biochemistry, University of Groningen Kerklaan 30,  9751 NN Haren, The Netherlands EMBO J., inpress Ras of complex proteins (Roc) belongs to the superfamily of Ras-related small  G-proteins that always occurs in tandem with the C-terminal of Roc (COR)  domain. This Roc–COR tandem is found in the bacterial and eukaryotic world.  Its most prominent member is the leucine-rich repeat kinase LRRK2, which  is mutated and activated in Parkinson patients. Here, we investigated  biochemically and structurally the Roco protein from Chlorobium tepidum.  We show that Roc is highly homologous to Ras, whereas the COR domain is a  dimerisation device. The juxtaposition of the G-domains and mutational analysis  suggest that the Roc GTPase reaction is stimulated and/or regulated by  dimerisation in a nucleotide-dependent manner. The region most conserved  between bacteria and man is the interface between Roc and COR, where  single-point Parkinson mutations of the Roc and COR domains are in close  proximity. The analogous mutations in C. tepidum Roc–COR decrease the  GTPase reaction rate, most likely due to a modification of the interaction  between the Roc and COR domains. Submitted by: Peter Van Haastert [P.J.M.van.Haastert@rug.nl] -------------------------------------------------------------------------------- Heat shock protein 90 regulates development in dictyostelium discoideum Ritwick Sawarkar (2, #), Nainita Roy (1, #), Sanjana Rao (1), Swetha Raman (3),  Venketesh S (1),Suguna K (3) and Utpal Tatu(1) (1) Department of Biochemistry,  (2) Molecular Reproduction, Development and Genetics, (3) Molecular Biophysics Unit,  Indian Institute of Science, Bangalore 560 012, India # These authors contributed equally to the manuscript Journal of Molecular Biology (accepted) Cytosolic heat shock protein 90 (Hsp90) has been implicated in diverse  biological processes ranging from protein folding, cell cycle control, signal  transduction, development and morphological evolution. Available model  systems to study Hsp90 function either allow an ease of manipulation for  biochemical studies or facilitate phenomenological study of its role in  influencing phenotype. In this paper we have explored the use of the  cellular slime mold Dictyostelium discoideum to examine cellular functions  of Hsp90 in relation to its multicellular development. In addition to cloning,  purification, biochemical characterization and examination of its crystal  structure our studies using a pharmacological inhibitor of Hsp90 demonstrate  a role for the cytoplasmic isoform (HspD) in D. discoideum development.  Inhibition of HspD function using geldanamycin resulted in delayed aggregation  and an arrest of D. discoideum development at the ‘mound’ stage. Crystal  structure of the amino terminal domain of HspD showed a binding pocket similar  to that described for yeast Hsp90. Fluorescence spectroscopy as well as  geldanamycin coupled beads affinity-pull down confirmed a specific interaction  between HspD and geldanamycin. The results presented here provide an  important insights into the function of HspD in D. discoideum development and  emphasize the potential of the cellular slime mold to serve as an effective model  to study the many roles of Hsp90 at a cellular and an organismal level. Submitted by: Ritwick Sawarkar [ritwick@mrdg.iisc.ernet.in] ============================================================== [End dictyNews, volume 31, number 7]