dictyNews
Electronic Edition
Volume 38, number 13
May 11, 2012

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Abstracts
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Distinct Functional Roles of the two Terminal Halves of Eukaryotic 
Phosphofructokinase

Oscar H. Martínez-Costa, Valentina Sánchez, Antonio Lázaro, 
Eloy D. Hernández, Keith Tornheim and Juan J. Aragón


Biochemical Journal, published

Eukaryotic phosphofructokinase, a key regulatory enzyme in glycolysis, 
has homologous N- and C-terminal domains thought to result from 
duplication, fusion, and divergence of an ancestral prokaryotic gene. It 
has been suggested that both the active site and the fructose 2,6-P2 
allosteric site are formed by opposing N- and C-termini of subunits 
oriented antiparallel in a dimer. On the contrary, we show here that in 
fact the N-terminal halves form the active site, since expression of the 
N-terminal half of the enzymes from Dictyostelium discoideum and 
human muscle in phosphofructokinase-deficient yeast restored growth 
on glucose. However, the N-terminus alone was not stable in vitro. The 
C-terminus is not catalytic but is needed for stability of the enzyme, as 
is the connecting peptide that normally joins the two domains (here 
included in the N-terminus). Co-expression of homologous, but not 
heterologous, N- and C-termini yielded stable, fully active enzymes in 
vitro with sizes and kinetic properties similar to those of the wild type 
tetrameric enzymes. This indicates that the separately translated 
domains can fold sufficiently well to bind to each other, that such binding 
of complementary domains is stable and that the alignment is sufficiently 
accurate and tight as to preserve metabolite binding sites and allosteric 
interactions.


Submitted by Juan Aragón [[log in to unmask]]
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EGF-like peptide-enhanced cell movement in Dictyostelium is mediated 
by protein kinases and the activity of several cytoskeletal proteins

Robert J. Huber a and Danton H. O'Day a,b

a Department of Cell and Systems Biology, University of Toronto, 25 
Harbord st., Toronto, Ontario, Canada, M5S 3G5
b Department of Biology, University of Toronto at Mississauga, 3359 
Mississauga rd. N., Mississauga, Ontario, Canada, L5L 1C6
 
 
Cellular Signalling, in press

DdEGFL1, a synthetic Epidermal Growth Factor-Like (EGFL) peptide 
based on the first EGFL repeat of the extracellular matrix, cysteine-rich, 
calmodulin-binding protein CyrA, has previously been shown to sustain 
the threonine phosphorylation of a 210 kDa protein during the starvation 
of Dictyostelium cells. Immunoprecipitation coupled with a LC/MS/MS 
analysis identified the 210 kDa protein as vinculin B (VinB). VinB shares 
sequence similarity with mammalian vinculin, a protein that links the actin 
cytoskeleton to the plasma membrane. Both threonine phosphorylated 
VinB (P-VinB) and VinB-GFP localized to the cytoplasm and cytoskeleton 
of Dictyostelium amoebae. VinB-GFP was also shown to be threonine 
phosphorylated and co-immunoprecipitated with established 
vinculin-binding cytoskeletal proteins (e.g. myosin II heavy chain, actin, 
alpha-actinin, talin). P-VinB and VinB-GFP were detected in DdEGFL1 
pull-down assays, which also identified a 135 kDa phosphothreonine 
protein and two phosphotyrosine proteins (35 and 32 kDa) as potential 
components of the DdEGFL1 signalling pathway. DdEGFL1-enhanced 
cell movement required the cytoskeletal proteins talin B and paxillin B 
and tyrosine kinase activity mediated by PKA signalling, however VinB 
threonine phosphorylation was shown to be independent of PI3K/PLA2 
signalling and PI3K and PKA kinase activity. Finally, VinB-GFP 
over-expression suppressed DdEGFL1-enhanced random cell movement, 
but not folic acid-mediated chemotaxis. Together, this study provides the 
first evidence for VinB function plus new insight into the signalling 
pathway(s) mediating EGFL repeat/peptide-enhanced cell movement in 
Dictyostelium. This information is integrated into an emerging model that 
summarizes existing knowledge. 


Submitted by Danton H. O'Day [[log in to unmask]]
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Nucleoplasmic/nucleolar translocation and identification of a nuclear 
localization signal (NLS) in Dictyostelium BAF60a/SMARCD1 
homologue Snf12

Andrew Catalano a, and Danton H. O'Day a,b*

a Department of Cell and Systems Biology, University of Toronto, 
25 Harbord st., Toronto, Ontario, Canada, M5S 3G5
b Department of Biology, University of Toronto at Mississauga, 
3359 Mississauga rd. N., Mississauga, Ontario, Canada, L5L 1C6


Histochemistry and Cell Biology, in press

Dictyostelium is a model eukaryote for the study of several cellular 
processes however comparatively little is known about its nucleolus. 
Identification of nucleolar proteins is key to understanding this nuclear 
subcompartment but only four have been identified in Dictyostelium. 
As discussed in this article, a potential relationship between nucleolar 
NumA1 and BAF60a/SMARCD1 suggested BAF60a may also reside 
in the nucleolus. Here we identify BAF60a homologue Snf12 as the fifth 
nucleolar protein in Dictyostelium. Immunolocalization experiments 
demonstrate that Snf12 is nucleoplasmic but translocates to nucleoli 
upon actinomycin-D-induced transcription inhibition (0.05 mg/mL, 
4 hours). Translocation was accompanied by a microtubule-independent 
protrusion of nucleolar Snf12 regions from the nucleus followed by 
detection of Snf12 in cytoplasmic circles for at least 48 hours. Residues 
372KRKR375 are both necessary and sufficient for nucleoplasmic 
localization of Snf12 and represent a functional nuclear localization 
signal (NLS), similar to recently identified NLSs in other Dictyostelium 
proteins. Since nucleolar and nucleoplasmic proteins redistribute during 
mitosis, we investigated Snf12 dynamics during this time. Dictyostelium 
undergoes closed mitosis, meaning its nuclear envelope remains intact. 
Despite this, during metaphase and anaphase Snf12 redistributed 
throughout the cytoplasm before reaccumulating in the nucleus during 
telophase, unlike the previously reported nucleoplasmic redistribution of 
nucleolar NumA1. The nuclear exit of Snf12 was independent of its 
putative nuclear export signal and not inhibited by exportin inhibition 
suggesting the redistribution of nuclear proteins during mitosis in 
Dictyostelium is mediated by other mechanisms. Snf12 is the second 
Dictyostelium nucleolar protein for which its dynamics during mitosis 
have been investigated.


Submitted by Danton H. O'Day [[log in to unmask]]
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[End dictyNews, volume 38, number 13]