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.
Eichinger
L
L
Adolf-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universität, Schillerstrasse 42, 80336 München, Germany.
Bähler
M
M
Dietz
M
M
Eckerskorn
C
C
Schleicher
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D020930:MAP Kinase Kinase Kinases; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015800:Protozoan Proteins; D011817:Rabbits; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D015800:Protozoan Proteins; D029701:Saccharomyces cerevisiae Proteins; C036571:severin protein, Dictyostelium; C112607:severin kinase, Dictyostelium; D017346:Protein Serine-Threonine Kinases; D020930:MAP Kinase Kinase Kinases; C078754:STE20 protein, S cerevisiae