Characterization and Cloning of a DictyosteliumSte20-like Protein Kinase That Phosphorylates the Actin-binding Protein Severin*
- From the ‡Adolf-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universität, Schillerstrasse 42, 80336 München, Germany and the §Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany
Abstract
After receiving an external stimulusDictyostelium 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 inDictyostelium suggests a direct signal transduction from the plasma membrane to the cytoskeleton by phosphorylation of actin-binding proteins.
Footnotes
-
↵* This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Friedrich Baur Stiftung, the Fonds der Chemischen Industrie, and the European Union (to M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF059534.
-
↵¶ To whom correspondence should be addressed: Adolf-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universität München, Schillerstr. 42, 80336 München, Germany. Tel.: 49-89-5996876; Fax: 49-89-5996882; E-mail: schleicher{at}bio.med.uni-muenchen.de.
-
↵1 The abbreviations used are: MAPK, mitogen-activated protein kinase; PAK, p21-activated protein kinase; GCK, germinal center kinase; SOK, STE20/oxidant stress response kinase; MES, 2-(N-morpholino)ethane sulfonic acid; Krs, kinase responsive to stress; BLAST, basic local alignment search tool; MIHCK, myosin I heavy chain kinase; Rab8ip, rab8 interacting protein; MST, mammalian sterile 20-like; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; GTPγS, guanosine 5′-3-O-(thio)triphosphate; PCR, polymerase chain reaction; kb, kilobase pair(s); aa, amino acid(s).
-
- Received November 18, 1997.
- Revision received February 25, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
