Ste20-like kinases and regulator proteins in the cytoskeleton of Dictyostelium discoideum

The cytoskeleton in most eukaryotes consists of actin filaments,
intermediate filaments, microtubules and specific associated
proteins. It determines the shape and the polarity of a cell and is
inevitable for the coordination of cell movement. The regulation of
this complex structure requires a highly organised and specialised
signalling network. Ste20-like kinases and the regulator protein
Mo25 (Morula protein 25) are part of this signalling network. The
main objective of this work was the functional characterisation of
the regulator protein Mo25 and the Ste20-like kinases Fray1, Fray2
(Frayed kinase 1/2) and DstC (Dictyostelium serine threonine kinase
C) in the amoeba Dictyostelium discoideum (D. discoideum). An
additional project was to map and characterise the actin and actin
related genes in the genome of the fresh water foraminifer
Reticulomyxa filosa (R. filosa). Mo25 is a highly conserved 40 kDa
scaffolding protein with a 60% identity from amoeba to man. The
disruption of the mo25 gene in D. discoideum results in very large,
multinucleated cells which are unable to complete cytokinesis.
Growth as well as development is severely delayed in the Mo25-minus
strain. Furthermore, in phototaxis assays performed with
multicellular aggregates (slugs), the Mo25-minus slugs were unable
to migrate towards the light source. These findings imply that Mo25
plays an important role in cytokinesis, growth and cell polarity.
We could link the Ste 20-like kinase SvkA (severin kinase), a
homolog of the human Mst3, Mst4 (Mammalian Ste20-like kinase 3/4)
and Ysk1/Sok1 (Yeast Sps1/Ste20-related kinase 1, Suppressor of
Kinase 1) kinases to Mo25 as a binding partner. To further
elucidate the interaction of Mo25 with SvkA as well as their role
in cytokinesis or polarity signalling, we generated a series of
GFP–Mo25 rescue constructs with distinct point mutations in
protein-protein interaction surfaces and transformed these into the
Mo25-minus background. The kinase domains of the Ste20-like
kinases, Fray1 and Fray2 in D. discoideum are highly homologous to
the catalytic domains of OSR1 (Oxidative stress response kinase 1)
and SPAK (Ste20/SPS1-related proline-alanine-rich protein kinase)
in humans and Frayed in fruit fly. Here, we generated the knockout
clones Fray1-minus, Fray2-minus, and the double knockout
Fray2Fray1-minus in D. discoideum. In developmental studies,
Fray2-minus did not show an altered phenotype, whereas Fray1-minus
and Fray2Fray1-minus developed slightly slower into fruiting
bodies. When grown in shaking culture, Fray1-minus and
Fray2Fray1-minus showed a reduced growth rate compared to
Fray2-minus and the wild type. In addition, by using a GFP-Trap
resin we identified a binding partner of Fray1, a yet unknown
protein that we named FRIP (Fray Interacting Protein). FRIP mainly
consists of a CBS (Cystathionine beta synthase) domain pair and is
30% identical to the gamma subunit of the AMPK (5‘ adenosine mono
phosphate-activated protein kinase) complex in D. discoideum. The
Ste20-like kinase DstC has been described to be a regulator of the
actin driven process of phagocytosis. The catalytic domain of DstC
is most similar to the mammalian kinase Mst2 (Mammalian Ste20-like
kinase 2) and Hippo (“Hippopotamus”-like phenotype) of D.
melanogaster. We could map the sorting signal that localises DstC
to phagocytic cups and acidic vesicles to about 90 amino acids.
Here we present an array of distinct point mutations for the
identification of the exact localisation signal. R. filosa is a
fresh water protist which belongs to the the group of Foraminifera
within the Rhizaria. The R. filosa genome is the first
foraminiferal and only the second rhizarian genome to be
deciphered. In this bioinformatics project, we could identify, map
and characterise four new actin genes in addition to the already
known actin and about 40 genes that code for potential actin
related proteins of seven different protein classes.