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dictyNews Volume 19 Number 08
Dicty News
Electronic Edition
Volume 19, number 8
October 12, 2002
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu.
Back issues of Dicty-News, the Dicty Reference database and other useful
information is available at DictyBase--http://dictybase.org.
==============================
Postdoc Position Available
==============================
Postdoctoral position in the Wellcome Trust Biocentre at the University
of Dundee
A postdoctoral position is available in Dr. Inke Nthke s laboratory to
join a team studying the function of the Adenomatous Polyposis Coli tumour
suppressor protein (APC). The project will involve using Dictyostelium
Discoideum to investigate the role of APC in cytoskeletal dynamics and
organisation. The general aim of work in the laboratory is to determine
how the diverse functions of APC relate to its role in cancer and how
these functions are co-ordinated and regulated. Experimental approaches
include general cell- and molecular biology techniques combined with
high-resolution fluorescence microscopy and use of Dictyostelium as a
model organism. The candidate will join a small, dynamic, highly
interactive research team with active collaborations worldwide.
A proven research ability and motivation are required and experience
in working with Dictyostelium is requisite. Experience in general
cell biology and molecular biology is essential. The position is
available immediately for 2 years in the first instance. For informal
enquiries, please contact Dr. Inke S. Nthke (+ 44 1382 345821;
e-mail: i.s.nathke@dundee.ac.uk). Applications should be directed to:
Janette Cordiner, School Administrator, School of Life Sciences,
MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH;
j.m.cordiner@dundee.ac.uk. Please cite reference SC/763/1 on the
application.
The Wellcome Trust Biocentre is part of the internationally renowned
WTB/MSI complex at the University of Dundee. The complex provides an
extremely interactive research environment and houses nearly 400
research staff. Further information about research in the WTB/MSI
complex can be obtained at http://www.dundee.ac.uk/biocentre/.
=============
Abstracts
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CaMBOT: Profiling and Characterizing Calmodulin Binding Proteins
Danton H. O Day
Department of Zoology, University of Toronto at Mississauga, Mississauga,
ON L5L 1C6
CANADA
Cellular Signalling (in press)
Abstract
Calmodulin (CaM) is an essential calcium-binding protein that binds to and
activates a diverse population of downstream targets (calmodulin binding
proteins; CaMBPs) that carry out its critical signalling functions. In
spite of the central importance of CaM in Ca2+-mediated signal transduction
pathways in all eukaryotes, most CaMBPs remain to be identified and
characterized. SDS-PAGE followed by gel overlay with recombinant,
metabolically radiolabelled CaM (Calmodulin Binding Overlay Technique,
CaMBOT) is a valuable method for following behavioral, developmental,
forensic and physiological changes in total CaMBP populations and to
identify candidate CaMBPs for further study. CaMBOT has also been adapted
to isolate cDNAs encoding novel CaMBPs in various organisms. Recently the
method was used to examine the CaMBP complement encoded by the Arabidopsis
genome and to identify a new family of transcription activators. To add to
its diversity, CaMBOT may be useful for finding target proteins for work
on phytoremediation and for the screening of pharmaceuticals and toxic
agents that, directly or indirectly, affect CaM and its target proteins.
This review discusses all of these topics and the role of CaMBOT in
characterizing a functional unit of the proteome proteins regulated by
calmodulin.
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Myosin Heavy Chain Kinase B participates in the regulation of myosin
assembly into the cytoskeleton.
Maribel Rico and Thomas T. Egelhoff
Department of Physiology and Biophysics,
Case Western Reserve University, Cleveland. OH. 44106-4970
Journal of Cellular Biochemistry, in press
Abstract
Myosin II plays critical roles in events such as cytokinesis, chemotactic
migration, and morphological changes during multicellular development. The
amoeba Dictyostelium discoideum provides a simple system for the study of
this contractile protein. In this system, myosin II filament assembly is
regulated by myosin heavy chain (MHC) phosphorylation in the tail region of
the molecule. Earlier studies identified an alpha- kinase, myosin heavy
chain kinase A (MHCK A), which phosphorylates three mapped threonine
residues in the myosin tail, driving myosin disassembly. Using molecular
and genomic approaches we have identified a series of related kinases in
Dictyostelium. The enzyme MHCK B shares with MHCK A a domain organization
that includes a highly novel catalytic domain coupled to a
carboxyl-terminal WD repeat domain.
We have engineered, expressed and purified a FLAG-tagged version of the
novel kinase. In the present study, we report detailed biochemical and
cellular studies documenting that MHCK B plays a physiological role in the
control of Dictyostelium myosin II assembly and disassembly during the
vegetative life of Dictyostelium amoebae. The presented data supports a
model of multiple related MHCKs in this system, with different regulatory
mechanisms and pathways controlling each enzyme.
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Dictyostelium discoideum has a single diacylglycerol kinase gene with
similarity to mammalian theta isoforms
Marc A. de la Roche1, Janet L. Smith2, Maribel Rico3, Silvia Carrasco4,
Isabel Merida4, Lucila Licate3, Graham P. Ct1, and Thomas T. Egelhoff3#
1. Department of Biochemistry
Queen s University
Kingston, Ontario K7L 3N6, Canada
2. Boston Biomedical Research Institute
64 Grove St.
Watertown, MA 02472-2829
3. Department of Physiology and Biophysics
Case Western Reserve School of Medicine
Cleveland, OH 44016-4970
4 Department of Immunology and Oncology
National Center for Biotechnology
Campus de Cantoblanco
Madrid 28049, Spain
# Corresponding author
Biochemical Journal (online as preprint), in press
Synopsis
Diacylglycerol kinases (DGKs) phosphorylate the neutral lipid
diacylglycerol (DG) to produce phosphatidic acid (PA). In mammalian
systems DGKs are a complex family of at least 9 isoforms that are thought
to participate in downregulation of DG-based signaling pathways and perhaps
activation of PA-stimulated signaling events. We report here that the
simple protozoan amoeba Dictyostelium discoideum appears to contain a
single gene encoding a DGK enzyme. This gene, dgkA, encodes a deduced
protein that contains three C1-type cysteine-rich repeats, a DGK catalytic
domain most closely related to the theta subtype of mammalian DGKs, and a
carboxyl-terminal segment containing a proline/glutamine-rich region and a
large aspargine-repeat region. This gene corresponds to a previously
reported myosin II heavy chain kinase designated "MHC-PKC", but our
analysis clearly demonstrate that this protein does not, as suggested by
earlier data, contain a protein kinase catalytic domain. A FLAG-tagged
version of DgkA expressed in Dictyostelium displayed robust diacylglycerol
kinase activity. Earlier studies indicating that disruption of this locus
alters myosin II assembly levels in Dictyostelium raise the intriguing
possibility that DG and/or PA metabolism may play a role in controlling
myosin II assembly in this system.
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A Bifunctional Di-Glycosyltransferase Forms the Fuca1,2Galb1,3-Disaccharide
on Skp1 in the Cytoplasm of Dictyostelium
Hanke van der Wel, Suzanne Z. Fisher, and Christopher M. West
Department of Anatomy and Cell Biology, University of Florida College of
Medicine, Gainesville, FL 32610-0235 USA
J. Biol. Chem., in press
Skp1 is a subunit of the SCF-family of E3-ubiquitin ligases and of other
regulatory complexes in the cytoplasm and nucleus. In Dictyostelium, Skp1
is modified by a pentasaccharide with the type I blood group H antigen
(Fuca1,2Galb1,3GlcNAc-) at its core. Addition of the Fuc is catalyzed by FT85,
a 768-amino acid protein whose fucosyltransferase activity maps to the
C-terminal half of the protein. A strain whose FT85 gene is interrupted by
a genetic insertion produces a truncated, GlcNAc-terminated glycan on Skp1,
suggesting that FT85 may also have b-galactosyltransferase activity. In
support of this model, highly-purified native and recombinant FT85 are each
able to galactosylate Skp1 from FT85-mutant cells. Site-directed mutagenesis
of predicted key amino acids in the N-terminal region of FT85 abolishes Skp1
b-galactosyltransferase activity with minimal effects on the fucosyltransferase.
In addition, a recombinant form of the N-terminal region exhibits
b-galactosyltransferase but not fucosyltransferase activity. Kinetic
analysis of FT85 suggests that its two glycosyltransferase activities
normally modify Skp1 processively but can have partial function individually.
In conclusion, FT85 is a bifunctional di-glycosyltransferase that appears to
be designed to efficiently extend the Skp1 glycan in vivo.
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Evolutionary and functional implications of the complex glycosylation of Skp1,
a cytoplasmic/nuclear glycoprotein associated with polyubiquitination
Christopher M. West
Department of Anatomy and Cell Biology, University of Florida College of
Medicine, Gainesville, FL 32610-0235 (USA), Fax +1 352 392 3305, e-mail:
westcm@ufl.edu
Cell and Molecular Life Sciences, in press.
Protein degradation is regulatory for the cell cycle, signal transduction and
gene transcription. A critical step is the selective marking of the target
protein, resulting in polyubiquitination by one of a large number of E3-ubiquitin
ligases. Both target marking and E3-ubiquitin ligase activity are associated
with common as well as unusual posttranslational modifications. For example,
hydroxylation of Pro-residues and modification of Asn-residues by high-mannose
sugar chains can target the modified proteins for rapid polyubiquitination
in the mammalian cytoplasm. Both prolyl hydroxylation and glycosylation also
occur on Skp1, a subunit of the SCF-class of E3-ubiquitin ligases, from
Dictyostelium. In this case, a pentasaccharide containing Gal, Fuc and GlcNAc
is attached to the HyPro-residue. The sugars are added sequentially by enzymes
that reside in the cytoplasm rather than the secretory pathway. Two of the
glycosyltransferases appear to be positioned in ancient evolutionary lineages
that bridge prokaryotes and eukaryotes. The first, which attaches GlcNAc to
HyPro, is related to enzymes that form a-GalNAc- and a-GlcNAc-Ser/Thr linkages
in the Golgi. GlcNAc is extended by a bifunctional glycosyltransferase that
mediates the ordered addition of b1,3-linked Gal and a1,2-linked Fuc, using
an architecture resembling that of 2-domain prokaryotic glycosyltransferases
involved in glycosaminoglycan synthesis. Mutational and pharmacological
perturbation of glycosylation alters the subcellular localization of Skp1 and
growth properties of cells. Prolyl hydroxylation and complex O-glycosylation
provide the cell with new options for epigenetic regulation of protein turnover
in its cytoplasmic and nuclear compartments.
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Molecular Cloning and Expression of a UDP-GlcNAc:Hydroxyproline Polypeptide
GlcNAc-Transferase that Modifies Skp1 in the Cytoplasm of Dictyostelium
Hanke van der Wela, Howard R. Morrisb,c, Maria Panicob, Thanai Paxtonb, Anne
Dellb, Lee Kaplana, and Christopher M. Westa,d
aDepartment of Anatomy & Cell Biology, University of Florida College of
Medicine, Gainesville, FL USA 32610-0235; bDepartment of Biological
Sciences, Imperial College of Science, Technology and Medicine, London
SW7 2AY, UK; cM-SCAN Research and Training Center, Silwood Park,
Ascot SL5 7PZ, UK
J. Biol. Chem., in press.
Skp1 is a ubiquitous eukaryotic protein found in several cytoplasmic and
nuclear protein complexes, including the SCF-type E3 ubiquitin ligase. In
Dictyostelium, Skp1 is hydroxylated at proline-143 which is then modified
by a pentasaccharide chain. The enzyme activity that attaches the first
sugar, GlcNAc, was previously shown to copurify with the GnT51 polypeptide
whose gene has now been cloned using a proteomics approach based on a Q-TOF
hybrid mass spectrometer. When expressed in E. coli, recombinant GnT51
exhibits UDP-GlcNAc:hydroxyproline Skp1 GlcNAc-Transferase activity. Based
on amino acid sequence alignments, GnT51 defines a new family of microbial
polypeptide glycosyltransferases that appear to be distantly related to the
catalytic domain of mucin-type UDP-GalNAc:Ser/Thr polypeptide
a-GalNAc-Transferases expressed in the Golgi compartment of animal cells.
This relationship is supported by the effects of site-directed mutagenesis
of amino acids associated with GnT51's predicted DxD-like motif, DAH. In
contrast, GnT51 lacks the NH2-terminal signal anchor sequence present in
the Golgi enzymes, consistent with the cytoplasmic localization of the Skp1
acceptor substrate and the biochemical properties of the enzyme. The first
glycosylation step of Dictyostelium Skp1 is concluded to be mechanistically
similar to that of animal mucin type O-linked glycosylation except that it
occurs in the cytoplasm rather than the Golgi compartment of the cell.
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GenePath: a System for Automated Construction of Genetic Networks from Mutant
Data
Blaz Zupan, Janez Demsar, Ivan Bratko, Peter Juvan, John A. Halter, Adam
Kuspa and Gad Shaulsky
University of Ljubljana, Faculty of Computer and Information Science and
Jozef Stefan Institute, Ljubljana, Slovenia
Departments of PM&R and Division of Neuroscience, Biochemistry and Molecular
Biology, Molecular and Human Genetics, Baylor College of Medicine, Houston,
TX 77030, USA
Bioinformatics, in press
ABSTRACT
Motivation: Genetic networks are often used in the analysis of biological
phenomena. In classical genetics, they are constructed manually from
experimental data on mutants. The field lacks formalism to guide such
analysis, and accounting for all the data becomes complicated when large
amounts of data are considered.
Results: We have developed GenePath, an intelligent assistant that automates
the analysis of genetic data. GenePath employs expert-defined patterns to
uncover gene relations from the data, and uses these relations as constraints
in the search for a plausible genetic network. GenePath formalizes genetic
data analysis, facilitates the consideration of all the available data in a
consistent manner, and the examination of the large number of possible
consequences of planned experiments. It also provides an explanation
mechanism that traces every finding to the pertinent data.
Availability: GenePath can be accessed at http://genepath.org.
Contact: gadi@bcm.tmc.edu
Supplementary information: Supplementary material is available at
http://genepath.org/bi-supp.
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Tail chimeras of Dictyostelium myosin II support cytokinesis and other
myosin II activities but not full development
Shi Shu1, Xiong Liu1, Carole A. Parent 2, Taro Q. P. Uyeda3, and Edward D.
Korn*1
1 Laboratory of Cell Biology, National Heart, Lung, and Blood Institute,
and 2 Laboratory of Cellular and Molecular Biology, National Cancer Institute,
National Institutes of Health, Bethesda, MD 20892 and 3Gene Function Research
Center, Tsukuba Central #4, National Institute of Advanced Industrial Science
and Technology (AIST), Higashi 1-1-1, Tsukuba, Ibaraki 305-8562, Japan
*Author for correspondence (e-mail: edk@nih.gov)
Short title: In vivo activities of myosin II tail chimeras
Key words: myosin II; cytokinesis; chemotaxis; development; Con A capping.
Journal of Cell Science, in press
SUMMARY
Dictyostelium lacking myosin II cannot grow in suspension culture, develop
beyond the mound stage or cap concanavalin A receptors and chemotaxis is
impaired. Recently, we showed that the actin-activated MgATPase activity
of myosin chimeras in which the tail domain of Dictyostelium myosin II heavy
chain is replaced by the tail domain of either Acanthamoeba or chicken
smooth muscle myosin II is unregulated and about 20 times higher than
wild-type myosin. The Acanthamoeba chimera forms short bipolar filaments
similar to, but shorter than, filaments of Dictyostelium myosin, and the
smooth muscle chimera forms much larger side-polar filaments. We now find
that the Acanthamoeba chimera expressed in myosin null cells localizes to
the periphery of vegetative amoeba similarly to wild-type myosin but the
smooth muscle chimera is heavily concentrated in a single cortical patch.
Despite their different tail sequences and filament structures and different
localization of the smooth muscle chimera in interphase cells, both chimeras
support growth in suspension culture and concanavalin A capping and co-
localize with the Con A cap but the Acanthamoeba chimera subsequently
disperses more slowly than wild-type myosin and the smooth muscle chimera
apparently not at all. Both chimeras also partially rescue chemotaxis.
However, neither supports full development. Thus, neither regulation of
myosin activity, nor regulation of myosin polymerization nor bipolar
filaments is required for many functions of Dictyostelium myosin II and
there may be no specific sequence required for localization of myosin to
the cleavage furrow.
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Macromolecular Architecture in Eukaryotic Cells Visualized by Cryo-Electron
Tomography
Ohad Medalia, Igor Weber, Achilleas S. Frangakis, Guenther Gerisch
and Wolfgang Baumeister
Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany
Science, in press.
Abstract
Electron tomography of vitrified cells is a non-invasive 3-dimensional
imaging technique which opens up new vistas for exploring the supramolecular
organization of the cytoplasm. We applied this technique to Dictyostelium
cells focusing on the actin cytoskeleton. In actin networks reconstructed
without prior removal of membranes or extraction of soluble proteins, the
crosslinking of individual microfilaments, their branching angles and
membrane attachment sites can be analyzed. At a resolution of 5 to 6 nm,
single macromolecules with distinct shapes, such as the 26S proteasome,
can be identified in an unperturbed cellular environment.
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Purification and Renaturation of Dictyostelium Recombinant Alkaline
Phosphatase by Continuous Elution Electrophoresis
Muatasem Ubeidat and Charles L. Rutherford
Biology Department, Molecular and Cellular Biology Section, Virginia
Polytechnic Institute and State University, Blacksburg, VA 24061-0406
Protein Expression and Purification, In Press
Abstract
A 1583 bp fragment of Dictyostelium alp cDNA (94% of the gene) was cloned in
pET32a+. The enzyme was expressed in an inactive form in the inclusion body
of the expression host BL21-CodonPlus"!(DE3)-RIL. The recombinant ALP
constituted more than 50% of the total protein in the inclusion body and
25-30% of the total protein in the expression host after 3 h induction with
IPTG at 37 C. A continuous elution polyacrylamide gel electrophoresis
procedure was used to purify the recombinant enzyme. This technique
yielded a homogenous protein that retained enzymatic activity after dialysis
without further treatment. A yield of 5 mg per liter of culture broth was
obtained with a specific activity of approximately 0.7 nmol/min/mg of
protein (0.7 mU/mg). Immunoinhibition studies using a polyclonal antibody
produced against the recombinant protein showed complete inhibition of
enzymatic activity when the enzyme was preincubated with the antibody at a
1: 1000 dilution. The enzyme exhibited a pH optimum of approximately 9.0.
The substrate specificity indicated that the Dictyostelium enzyme is a
typical broad range alkaline phosphatase.
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[End Dicty News, volume 19, number 8]
