Cloning and characterisation of a Dictyostelium STE20-like protein kinase DST2

In this study a new Dictyostelium STE20-like protein kinase DST2
(Dictyostelium STE20-like kinase 2) was cloned and characterised.
STE20 (Sterile 20) kinase was first identified in yeast as a
pheromone-induced serine/threonine protein kinase that acts
upstream of a MAP kinase cascade. Based on the domain structure,
DST2 belongs to the GCK subfamily of STE20-like protein kinases,
which include the mammalian STE20-like kinases (MST1/2/3), oxidant
stress response kinase SOK-1, and DST1 in Dictyostelium discoideum
which phosphorylates severin, a gelsolin-like F-actin fragmenting
protein. DST2 was discovered by screening of the D. discoideum cDNA
project database using DST1 as query. To confirm the existence of
the DST2 gene and its expression, Southern, Northern and Western
analyses of DST2 were carried out. It revealed that DST2 may have
two copies in the Dictyostelium genome and that DST2 was expressed
during all stages of D. discoideum development. In vitro kinase
assays with bacterially expressed fusion protein of full length
DST2 (aa461), the catalytic domain (aa287) and the regulatory
domain (aa174) showed that autophosphorylation of DST2 occurson the
regulatory domain and phosphorylates severin in the presence of a
Mn2+ or Mg2+. Purified catalytic domain of PKA phosphorylated the
regulatory domain of DST2 and caused an increase in the basal
autophosphorylation activity of DST2, suggesting that PKA may be a
potential upstream kinase of DST2 through the phosphorylation of
its regulatory domain. To understand the function of the
non-catalytic domain of DST2, three C-terminal truncation
constructs (aa1-421, aa1-368 and aa1-326) were used in comparison
to full length DST2 in in vitro kinase assays. Deletion of
C-terminal regions revealed an inhibitory region amino acids
326-461 of DST2. Gel filtration chromatography showed that DST2 was
eluted in a broad peak ranging from approximately 63 kDa to 400
kDa, suggesting that DST2 may exist in vivo as a monomer as well as
a high molecular weight complex. The influence of phosphorylated
and unphosphorylated severin on F-actin solutions was investigated
using falling-ball viscometry and fluorescence spectroscopy. It
turned out that phosphorylation by DST2 inhibits the F-actin
fragmenting activity of severin, suggesting that DST2 may be
directly involved in actin-cytoskeleton rearrangements.