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dictyNews Volume 31 Number 07
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.
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Abstracts
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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]
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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]
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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]
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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]
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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]
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[End dictyNews, volume 31, number 7]
