CHARACTERIZATION AND CLONING OF A DICTYOSTELIUM STE20-LIKE PROTEIN-KINASE THAT PHOSPHORYLATES THE ACTIN-BINDING PROTEIN SEVERIN

After receiving an external stimulus Dictyostelium amoebae are able to rearrange their actin cytoskeleton within seconds, and phosphorylatio n is a prime candidate for quick modification of cytoskeletal componen ts. We isolated a kinase from cytosolic extracts that specifically pho sphorylated severin, a Ca2+-dependent F-actin fragmenting protein. In gel filtration chromatography severin kinase eluted with a molecular m ass of about 300 kDa and contained a 62-kDa component whose autophosph orylation caused a mobility shift in SDS-polyacrylamide gel electropho resis and stimulated phosphorylation of severin. Severin kinase activi ty could be specifically precipitated with antibodies raised against t he 62-kDa polypeptide. Phosphorylation of severin was strongly reduced in the presence of Ca2+, indicating additional regulation at the subs trate level. Peptide sequencing and cloning of the cDNA demonstrated t hat the 62-kDa protein belongs to the Ste20p- or pal-activated protein kinase family. It is most closely related to the germinal center kina se subfamily with its N-terminal positioned catalytic domain followed by a presumptive regulatory domain at the C terminus. The presence of a Ste20-like severin kinase in Dictyostelium suggests a direct signal transduction from the plasma membrane to the cytoskeleton by phosphory lation of actin-binding proteins.