dictyNews Volume 26 Number 13

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dictyNews 
Electronic Edition 
Volume 26, number 13 
May 5, 2006 

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. 

Back issues of dictyNews, the Dicty Reference database and other  
useful information is available at dictyBase - http://dictybase.org. 

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

 

EppA, a putative substrate of DdERK2, regulates cAMP relay and chemotaxis  
in Dictyostelium discoideum  

Songyang Chen and Jeffrey E. Segall* 
Department of Anatomy and Structural Biology, Albert Einstein College of  
Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 

 
Eukaryotic Cell, in press 

The MAP kinase DdERK2 is critical for cAMP relay and chemotaxis to cAMP and  
folate, but the details downstream of DdERK2 are unclear. To search for  
targets of DdERK2 in Dictyostelium, 32PO43--labeled protein samples from  
wild type and Dderk2- cells were resolved by 2 dimensional electrophoresis.  
Mass spectrometry was used to identify a novel 45kD protein as a substrate  
of DdERK2 in Dictyostelium named EppA (ERK2-dependent phosphoprotein A).  
Mutation of potential DdERK2 phosphorylation sites demonstrated that  
phosphorylation on serine 250 of EppA is DdERK2-dependent. Changing  
serine 250 to alanine delayed development of Dictyostelium and reduced  
Dictyostelium chemotaxis to cAMP. Although overexpression of EppA had no  
significant effect on development or chemotaxis of Dictyostelium, disruption 
of the eppA gene led to delayed development and reduced chemotactic  
responses to both cAMP and folate. Both eppA gene disruption and  
overexpression of EppA carrying the serine 250 to alanine mutation led to  
inhibition of intracellular cAMP accumulation in response to chemoattractant 
cAMP, a pivotal process in Dictyostelium chemotaxis and development. Our  
studies indicate that EppA regulates extracellular cAMP induced signal  
relay and chemotaxis of Dictyostelium. 

 
Submitted by: Jeffrey E. Segall [segall@aecom.yu.edu]  
----------------------------------------------------------------------------- 

 
A cis-acting site controlling bidirectional transcription at the  
growth-differentiation transition in Dictyostelium  

Shigenori Hirose, Samuel H. Payne and William F. Loomis 

Cell and Developmetal Biology, Division of Biological Sciences, 
University of California San Diego, La Jolla, CA 92093-0368, USA 

 
Eukary. Cell, in press 

A pair of adjacent genes, impA and dia1, are divergently transcribed but  
expressed at different stages in the life cycle of Dictyostelium discoideum.  
The intervening 654 bp region carries cis-acting regions that are essential  
for transcription in both directions as well as repression of dia1 in  
growing cells. We have focused on a 112 bp region proximal to dia1 that is  
essential for bidirectional transcription. Analyses of a set of internal  
deletions showed that the sequence between positions 80 and 97  
(TTTGAATTTTTTGAATTT) is critical and that bases outside this region are  
dispensable. Site directed mutations within this critical region confirmed  
the importance of this sequence for transcription both to the right and to  
the left.  However, insertions of either 6 or 24 Ts into the run of 6 Ts  
separating the repeated GAA sequence had little effect on the functioning  
of the site in either direction suggesting that factors recognize the half  
sites TTGAATT separately. Inversion of the bases between positions 80 and 97  
greatly reduced expression in both directions indicating that orientation is  
critical for expression of both the near-by impA gene and the distal dia1  
gene which is more than 500 bp away. Comparison of 38 mutant constructs with  
multiple random variations in the region indicated that transcription  
factors may bind to a range of related sequences and still retain function.  
All functional constructs directed transcription both leftward and rightward  
while all non-functional constructs were impaired for transcription in both  
directions. It appears that the same transcription complex controls  
transcription of both impA and dia1. 

 
Submitted by: Bill Loomis [wloomis@ucsd.edu] 
----------------------------------------------------------------------------- 

 
Filamins: promiscuous organizers of the cytoskeleton  

 
Grzegorz M. Popowicz1, Michael Schleicher2, Angelika Noegel3, and  
Tad A. Holak1 

1 Max-Planck Institut fŸr Biochemie, 82152 Martinsried, Germany 
2 Institut fŸr Zellbiologie der LMU, 80336 MŸnchen, Germany 
3 Institut fŸr Biochemie I, Zentrum Molekulare Medizin Kšln, Medizinische  
FakultŠt, Univ. zu Kšln, 50931 Kšln, Germany 

 
Trends in Biochemical Sciences, in press 

Filamins are F actin cross-linking proteins. They are elongated homodimers;  
each monomer chain comprises an actin-binding domain and a rod segment which  
consists of six (Dictyostelium filamin) up to 24 (human filamin) highly  
homologous repeats of 96 amino acid residues which adopt an  
immunoglobulin-like fold. Hinges in the rod segment and reversible unfolding  
of single repeats might be the structural basis for the intrinsic  
flexibility of the actin networks generated by filamins. There is a plethora  
of filamin binding proteins which in most cases bind along the rod repeats.  
This rather promiscuous behaviour renders a filamin a versatile scaffold  
between the actin network and finely tuned molecular cascades from the  
membrane to the cytoskeleton.   

 
Submitted by: Paul Steimle [noegel@uni-koeln.de] 
----------------------------------------------------------------------------- 

 
The function of ammonium transporter A in the initiation of culmination of  
development in Dictyostelium discoideum  

Charles K. Singleton*, Janet H. Kirsten, and Colin J. Dinsmore  

Department of Biological Sciences  
Vanderbilt University  
VU Station B 351634  
Nashville TN 37235-1634, USA  

 
Eukaryotic Cell, in press 

The histidine kinase DhkC controls a phosphorelay involved in regulating  
the slug versus culmination choice during the multicellular developmental  
program of Dictyostelium discoideum.   When the relay is active, slug  
migration is favored due to the activation of a cAMP phosphodiesterase and  
the resultant lowering of the intracellular and extracellular levels of  
cAMP.  Ammonia signaling represents one input into the DhkC phosphorelay,  
and previous studies indicated that the ammonium transporter C inhibits the  
relay in response to low ammonia levels. Evidence is presented that another  
member of the family of ammonium transporters, AmtA, also regulates the  
slug/culmination choice. Under standard conditions of development, the wild  
type strain requires a transitional period of 2 to 3 hours to go from  
fingers to culminants, with some slugs forming and migrating briefly prior  
to culmination. In contrast, amtA null cells, like cells that lack DhkC,  
possessed a transitional period of only 1 to 2 hours and rarely formed  
slugs. Disruption of amtA in an amtC null strain overcame the slugger  
phenotype of that strain and restored its ability to culminate. Strains  
lacking AmtA were insensitive to the ability of ammonia to promote and  
prolong slug migration. These findings lead to the proposal that AmtA  
functions in ammonia sensing as an activator of the DhkC phosphorelay in  
response to perceived high ammonia levels.  

Submitted by: Charles Singleton [charles.k.singleton@vanderbilt.edu]  
----------------------------------------------------------------------------- 

DdPDE4, a novel cAMP-specific Phosphodiesterase  
at the Surface of Dictyostelium Cells 

Sonya Bader, Arjan Kortholt*, Helena Snippe and Peter J.M. Van Haastert 

Department of Molecular Cell Biology, University of Groningen, Kerklaan 30,  
9751NN Haren, the Netherlands 

 
Journal of Biological Chemistry, in press 

Dictyostelium discoideum cells possess multiple cyclic nucleotide  
phosphodiesterases that belong either to class I enzymes that are present  
in all eukaryotes, or to the rare beta-lactamase class II. We describe here  
the identification and characterization of DdPDE4, the third class I enzyme  
of Dictyostelium. The deduced amino acid sequence predicts that DdPDE4 has a  
leader sequence, two transmembrane segments and an extracellular catalytic  
domain that exhibits a high degree of homology with human cAMP-specific  
PDE8. Expression of the catalytic domain of DdPDE4 shows that the enzyme  
is a cAMP-specific phosphodiesterase activity with a KM of 10 “M;  cGMP is  
hydrolyzed at least 100-fold more slowly. The full length protein is shown  
to be membrane-bound with catalytic activity exposed to the extracellular  
medium. Northern blots and activity measurements reveal that expression of  
DdPDE4 is low during single cell stages, and increases at nine hours of  
starvation, corresponding with mound stage. A function during multicellular 
development is confirmed by the phenotype of ddpde4- knock-out strains,  
showing normal aggregation but impaired development from the mound stage on. 
These results demonstrate that DdPDE4 is a unique membrane-bound  
phosphodiesterase with an extracellular catalytic domain regulating  
inter-cellular cAMP during multicellular development.  

 
Submitted by: Peter Van Haastert [p.j.m.van.haastert@rug.nl] 
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[End dictyNews, volume 26, number 13]