Characterization of the Ste20-like kinase Krs1 of Dictyostelium discoideum.

Ste20-like kinases constitute a ubiquitous and expanding group of serine/threonine kinases, homologous to Ste20 in Saccharomyces cerevisiae. The social amoeba Dictyostelium discoideum contains at least 17 members of this kinase family, 13 from the germinal center kinase (GCK) subgroup and 4 p21-activated kinases (PAK). Here, we describe the kinase Krs1 which is encoded by the gene krsA, and phylogenetic analysis groups it into subfamily GCK-II together with human MST2 and MST1 or Hippo from Drosophila melanogaster. Significant similarities are found especially in the catalytic domain and in a short regulatory region (SARAH) which is thought to be important for protein/protein interactions. Northern blot analysis showed a single krsA transcript throughout development with an upregulation at 12h after the onset of starvation. The protein levels as detected with anti-Krs1 polyclonal antibodies revealed a similar pattern. Gel filtration experiments suggested that AX2 wild-type cells harbored multimeric forms of Krs1. In vitro phosphorylation assays with recombinant protein showed that the kinase exhibits autophosphorylation and accepts myelin basic protein and D. discoideum severin as substrates. A series of C-terminal deletions of Krs1 indicated that the regulatory domain in the C-terminal half contains inhibitory elements, and highlighted the importance of two predicted alpha-helices following subdomain XI of the classical catalytic domain. GFP-Krs1-overexpressing wild-type cells showed an enrichment of the kinase in the cortex, and motility of these cells during aggregation was reduced. Krs1 knockout strains exhibited only subtle differences to wild-type cells which suggests a certain redundancy among Ste20-like kinases in D. discoideum.