Characterization of Vasodilator-Stimulated Phosphoprotein DdVASP, a third profilin isoform and Ste20-like kinases from Dictyostelium discoideum

The major goal of the project was the investigation of proteins
that regulate dynamic rearrangements of the actin cytoskeleton in
Dictyostelium discoideum amoebae. Among the proteins studied in
detail were (i) D. discoideum vasodilator stimulated phosphoprotein
DdVASP and a new profilin isoform as putative regulators of
filopodia formation, and (ii) the Ste20-like kinases Krs1 and
Severin-Kinase as members of signalling cascades towards the actin
cytoskeleton. Filopodia are bundles of actin filaments projecting
from the cell surface. They are found on a variety of cell types
and are needed among others, for cell adhesion, sensory and
exploratory functions. Filopodia are frequently found associated
with sheet-like arrays of actin filaments called lamellipodia and
membrane ruffles. The function of the VASP homolog from D.
discoideum in filopodia formation was studied using molecular,
biochemical and cell biological approaches. The protein sequence of
DdVASP shares a significant homology to the members from other
species. The protein harbours two Ena/VASP homology domains EVH1
and EVH2 separated by a polyproline stretch. The EVH2 domain is
characterised by a G-actin binding site (GAB), an F-actin binding
site (FAB) and a tetramerisation domain. As a tetramer the DdVASP
protein can nucleate actin polymerization and bundle actin
filaments. The in vitro nucleating activity of DdVASP is salt
dependent and its nucleating activity is completely abolished at
100 mM salt. However, the F-actin bundling activity as determined
by the low speed sedimentation assay was not disturbed. The ability
of DdVASP to influence the binding of capping protein to the
growing ends of the actin filaments was tested through elongation
of capped F-actin seeds and by depolymerization of capped filaments
upon dilution below the critical concentration of the barbed ends.
Results from both sets of experiments showed that DdVASP cannot
remove capping protein from the barbed ends. The D. discoideum
formin dDia2, which was previously reported to be essential for
filopodia formation could elongate the capped F-actin seeds. In
vitro biochemical data led to the conclusion that the bundling
activity of DdVASP is the essential in vivo function to stabilise
actin filaments in emerging filopodia. To test this hypothesis, a
DdVASP null mutant was isolated. As expected the mutant failed to
produce any filopodia. Expression of wild type DdVASP, but not
DdVASPFAB, rescued the phenotype suggesting the importance of the
bundling activity of DdVASP in filopodia formation. To confirm that
the data obtained with DdVASP were not species specific, key
biochemical functions of HsVASP were also tested. The results
indicated that VASPs are functionally well conserved throughout
evolution. During this study, a third profilin isoform, profilin
III, was further characterised. Specific interaction between
profilin III and DdVASP was discovered. Profilin III shares a
limited homology at the amino acid level with the other two and
well studied profilins. Polyclonal antibodies that recognise only
the profilin III isoform showed that in wild type cells profilin
III represents less than 1% of all profilins. This suggests a
distinct role for profilin III, because a low protein concentration
argues against an actin sequestering function. Immunolocalisation
studies showed profilin III in filopodia and enriched at their
tips. Cells lacking the profilin III protein show defects in cell
motility during chemotaxis. The second part of the project dealt
with the characterisation of two D. discoideum Germinal Centre
Kinases (GCK). The catalytic domain of Krs1 was found to be highly
homologous to the catalytic region of human MST1 and MST2 from the
GCK-II subfamily. The regulatory region harbours the putative
inhibitory domain (aa 330-379) and a possible multimerization
(SARAH) domain (aa 412-458) described for GCKs in higher organisms.
This SARAH region spans about 50 amino acid residues, is located at
the extreme carboxyl terminus and most likely forms an  - helical
coiled-coil motif. GFP-Krs1 overexpressing wild type cells showed
an enrichment of the kinase in the cell cortex, and motility of
these cells during aggregation was reduced. Krs1 knockout strains
exhibited only subtle differences to wild type cells. Severin
kinase is encoded by the gene svkA, and phylogenetic analysis
groups it into subfamily GCK-III, along with human MST3, MST4 and
YSK/SOK-1. Immunoblot analysis with polyclonal antibodies showed an
uniform expression level throughout development. Gene disruption of
svkA resulted in cells that had problems to divide both in
submerged or in shaking cultures. Though the motility and
chemotaxis of these cells remain unaltered compared to the wild
type cells, the movement of the multicellular slugs is disturbed.
In addition, development was delayed and the mutant formed aberrant
fruiting bodies.