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 phosphorylation is a prime candidate for quick modification of cytoskeletal components. We isolated a kinase from cytosolic extracts that specifically phosphorylated severin, a Ca2+-dependent F-actin fragmenting protein. In gel filtration chromatography severin kinase eluted with a molecular mass of about 300 kDa and contained a 62-kDa component whose autophosphorylation caused a mobility shift in SDS-polyacrylamide gel electrophoresis and stimulated phosphorylation of severin. Severin kinase activity could be specifically precipitated with antibodies raised against the 62-kDa polypeptide. Phosphorylation of severin was strongly reduced in the presence of Ca2+, indicating additional regulation at the substrate level. Peptide sequencing and cloning of the cDNA demonstrated that the 62-kDa protein belongs to the Ste20p- or p21-activated protein kinase family. It is most closely related to the germinal center kinase 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 phosphorylation of actin-binding proteins.