dictyNews Volume 38 Number 13

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

Please submit abstracts of your papers as soon as they have been 
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or by using the form at 
http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. 

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========= 
Abstracts 
========= 

 
Distinct Functional Roles of the two Terminal Halves of Eukaryotic  
Phosphofructokinase 

Oscar H. Mart’nez-Costa, Valentina S‡nchez, Antonio L‡zaro,  
Eloy D. Hern‡ndez, Keith Tornheim and Juan J. Arag—n 

 
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 Arag—n [juanjose.aragon@uam.es] 
-------------------------------------------------------------------------------------- 

 
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. OÕDay 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. OÕDay [danton.oday@utoronto.ca] 
-------------------------------------------------------------------------------------- 

 
Nucleoplasmic/nucleolar translocation and identification of a nuclear  
localization signal (NLS) in Dictyostelium BAF60a/SMARCD1  
homologue Snf12 

Andrew Catalano a, and Danton H. OÕDay 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. OÕDay [danton.oday@utoronto.ca] 
============================================================== 
[End dictyNews, volume 38, number 13]