dictyNews
Electronic Edition
Volume 37, number 10
October 21, 2011

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Abstracts
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Dictyostelium puromycin-sensitive aminopeptidase A is a nucleoplasmic 
nucleomorphin-binding protein that relocates to the cytoplasm during mitosis

Andrew Catalano a, Yekaterina Poloz 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

Nucleomorphin (NumA1) is a nucleolar/nucleoplasmic protein linked to cell 
cycle in Dictyostelium. It interacts with puromycin-sensitive aminopeptidase 
A (PsaA) which in other organisms is a Zn2+-metallopeptidase thought to be 
involved in cell cycle progression and is involved in several human diseases. 
Here, we have shown that Dictyostelium PsaA contains domains characteristic 
of the M1 family of Zn2+ metallopeptidases: a GAMEN motif and a Zn2+ binding 
domain. PsaA colocalized with NumA1 in the nucleoplasm in vegetative cells and 
was also present to a lesser extent in the cytoplasm. The same localization pattern 
was observed in cells from slugs however in fruiting bodies PsaA was only detected 
in spore nuclei. During mitosis PsaA redistributed mainly throughout the cytoplasm. 
It possesses a functional nuclear localization signal (680RKRF683) necessary for 
nuclear entry. To our knowledge this is the first nuclear localization signal 
identified in a Psa from any organism. Treatment with Ca2+ chelators or 
calmodulin antagonists indicated that neither Ca2+ nor calmodulin are involved 
in PsaA localization. These results are interpreted in terms of the inter-relationship 
between NumA1 and PsaA in cell function in Dictyostelium.


Submitted by Danton H. O'Day [[log in to unmask]]
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The cyclin-dependent kinase inhibitor roscovitine inhibits kinase activity, 
cell proliferation, multicellular development, and Cdk5 nuclear translocation 
in Dictyostelium discoideum

Robert J. Huber 1,2 and Danton H. O'Day *1,2

1Department of Cell and Systems Biology, University of Toronto, 
25 Harbord Street, Toronto, ON, Canada M5S 3G5
2Department of Biology, University of Toronto Mississauga, 
3359 Mississauga Road North, Mississauga, ON, Canada L5L 1C6


Journal of Cellular Biochemistry, in press

Roscovitine, a cyclin-dependent kinase (Cdk) inhibitor, inhibited kinase 
activity and the axenic growth of Dictyostelium discoideum at micromolar 
concentrations. Growth was almost fully rescued in 50 µM and ~50% rescued 
in 100 µM roscovitine-treated cultures by the over-expression of Cdk5-GFP. 
This supports the importance of Cdk5 function during cell proliferation in 
Dictyostelium and indicates that Cdk5 is a primary target of the drug. 
Roscovitine did not affect the expression of Cdk5 protein during axenic 
growth but did inhibit its nuclear translocation. This novel result suggests 
that the effects of roscovitine could be due in part to altering Cdk5 
translocation in other systems as well. Kinase activity was inhibited by 
roscovitine in assays using AX3 whole cell lysates, but not in assays using 
lysates from Cdk5-GFP over-expressing cells. At higher concentrations, 
roscovitine impaired slug and fruiting body formation. Fruiting bodies that 
did form were small and produced relatively fewer spores many of which 
were round. However roscovitine did not affect stalk cell differentiation. 
Together with previous findings, these data reveal that roscovitine inhibits 
Cdk5 during growth and as yet undefined Cdks during mid-late development. 


Submitted by Danton H. O'Day [[log in to unmask]]
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RpkA, a highly conserved GPCR with a lipid kinase domain, has a role in 
phagocytosis and anti-bacterial defense 

Tanja Y. Riyahi, Frederike Frese, Michael Steinert, Napoleon N. Omosigho, 
Gernot Gloeckner, Ludwig Eichinger, Benoit Orabi, Robin S. B. Williams, 
Angelika A. Noegel 


PLoS ONE

RpkA (Receptor phosphatidylinositol kinase A) is an unusual seven-helix 
transmembrane protein of Dictyostelium discoideum with a G protein coupled 
receptor (GPCR) signature and a C-terminal lipid kinase domain (GPCR-PIPK) 
predicted as a phosphatidylinositol-4-phosphate 5-kinase. RpkA-homologs are 
present in all so far sequenced Dictyostelidae as well as in several other 
lower eukaryotes like the oomycete Phytophthora, and in the Legionella host 
Acanthamoeba castellani. Here we show by immunofluorescence that RpkA l
ocalizes to endosomal membranes and is specifically recruited to phagosomes. 
RpkA interacts with the phagosomal protein complex V-ATPase as proteins of 
this complex co-precipitate with RpkA-GFP as well as with the GST-tagged PIPK 
domain of RpkA. Loss of RpkA leads to a defect in phagocytosis as measured by 
yeast particle uptake. The uptake of the pathogenic bacterium Legionella 
pneumophila was however unaltered whereas its intra-cellular replication was 
significantly enhanced in rpkA-. The difference between wild type and rpkA- was 
even more prominent when L. hackeliae was used. When we investigated the 
reason for the enhanced susceptibility for L. pneumophila of rpkA- we could not 
detect a difference in endosomal pH but rpkA- showed depletion of 
phosphoinositides (PIP and PIP2) when we compared metabolically labeled
phosphoinositides from wild type and rpkA-. Furthermore rpkA- exhibited reduced 
nitrogen starvation tolerance, an indicator for a reduced autophagy rate. Our results 
indicate that RpkA is a component of the defense system of D. discoideum as well 
as other lower eukaryotes. 


Submitted by Angelika Noegel [[log in to unmask]]
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Identification of a Eukaryotic Reductive Dechlorinase and Characterization 
of Its Mechanism of Action on Its Natural Substrate 

Francisco Velazquez,1,* Sew Yu Peak-Chew,1 Israel S. Fernandez,1 
Christopher S. Neumann,2 and Robert R. Kay1 
*Correspondence: [log in to unmask] 

1 Laboratory of Molecular Biology, Medical Research Council, 
Cambridge CB2 0QH, UK 
2 Department of Microbiology, University of Washington, 
Seattle, WA 98195, USA 


Chemistry and Biology, in press

Chlorinated compounds are important environmental pollutants whose 
biodegradation may be limited by inefficient dechlorinating enzymes. 
Dictyostelium amoebae produce a chlorinated alkyl phenone called DIF 
which induces stalk cell differentiation during their multicellular development. 
Here we describe the identification of DIF dechlorinase. DIF dechlorinase is 
active when expressed in bacteria, and activity is lost from Dictyostelium cells 
when its gene, drcA, is knocked out. It has a Km for DIF of 88 nM and Kcat of 
6.7 s1. DrcA is related to glutathione S-transferases, but with a key 
asparagine-to-cysteine substitution in the catalytic pocket. When this change 
is reversed, the enzyme reverts to a glutathione S-transferase, thus 
suggesting a catalytic mechanism. DrcA offers new possibilities for the 
rational design of bioremediation strategies. 


Submitted by Francisco Velazquez [[log in to unmask]]
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[End dictyNews, volume 37, number 10]