A NOVEL TYPE OF PROTEIN-KINASE PHOSPHORYLATES ACTIN IN THE ACTIN-FRAGMIN COMPLEX

Actin-fragmin kinase (AFK) from Physarum polycephalum specifically pho sphorylates actin in the EGTA-resistant 1:1 actin-fragmin complex. The cDNA deduced amino acid sequence reveals two major domains of similar to 35 kDa each that are separated by a hinge-like proline/serine-rich segment of 50 residues, Whereas the N-terminal domain does not show a ny significant similarity to protein sequences from databases, there a re six complete kelch repeats in the protein that comprise almost the entire C-terminal half of the molecule. To prove the intrinsic phospho rylation activity of AFK, full-length or partial cDNA fragments were e xpressed both in a reticulocyte lysate and in Escherichia coli. In bot h expression systems, we obtained specific actin phosphorylation and l ocated the catalytic domain in the N-terminal half. Interestingly, thi s region did not contain any of the known protein kinase consensus seq uences, The only known sequence motif present that could have been inv olved in nucleotide binding was a nearly perfect phosphate binding loo p (P-loop), However, introduction of two different point mutations int o this putative P-loop sequence did not alter the catalytic activity o f the kinase, which indicates an as yet unknown mechanism for phosphat e transfer, Our data suggest that AFK belongs to a new class of protei n kinases and that this actin phosphorylation might be the first examp le of a widely distributed novel type of regulation of the actin cytos keleton in non-muscle cells.