A novel type of protein kinase phosphorylates actin in the actin-fragmin complex.

Abstract

Actin‐fragmin kinase (AFK) from Physarum polycephalum specifically phosphorylates actin in the EGTA‐resistant 1:1 actin‐fragmin complex. The cDNA deduced amino acid sequence reveals two major domains of approximately 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 any significant similarity to protein sequences from databases, there are six complete kelch repeats in the protein that comprise almost the entire C‐terminal half of the molecule. To prove the intrinsic phosphorylation activity of AFK, full‐length or partial cDNA fragments were expressed both in a reticulocyte lysate and in Escherichia coli. In both expression systems, we obtained specific actin phosphorylation and located the catalytic domain in the N‐terminal half. Interestingly, this region did not contain any of the known protein kinase consensus sequences. The only known sequence motif present that could have been involved in nucleotide binding was a nearly perfect phosphate binding loop (P‐loop). However, introduction of two different point mutations into this putative P‐loop sequence did not alter the catalytic activity of the kinase, which indicates an as yet unknown mechanism for phosphate transfer. Our data suggest that AFK belongs to a new class of protein kinases and that this actin phosphorylation might be the first example of a widely distributed novel type of regulation of the actin cytoskeleton in non‐muscle cells.

Characterization of a Phosphoprotein whose mRNA is Regulated by the Mitogenic Pathways in Dog Thyroid Cells

“…In particular, the Bcr protein (a serine/threonine kinase) [33] and the human A6 tyrosine kinase [ 341 have unusual kinase domains. Furthermore, the hepatitis transactivator protein [35] and the kinase specific for the actin-fragmin complex [36] have been reported to have intrinsic kinase activity despite lacking a classical eukaryotic protein kinase sequence. The catalytic domain of the lipid kinase family, such as PtdIns 3-kinase, contains a COOHterminal region distantly related to the catalytic domain of the protein kinase superfamily, and containing motifs conserved in subdomains VIB (motif I) and VII (motif 11) of the protein kinase catalytic domain [37-391.…”

WD Repeat Domains Target Dictyostelium Myosin Heavy Chain Kinases by Binding Directly to Myosin Filaments

“…Phosphorylation Assays-All kinase assays were performed at 25°C in 10 mM TES, pH 7.0, 2 mM MgCl 2 , 0.5 mM dithiothreitol, and 0.5 mM [␥- 32 P]ATP (250 -400 Ci/mol), using recombinant kinases at 20 -80 nM, as noted for each experiment. The substrates used in this study were MH-1 peptide, GST-2029 (38 kDa), and myosin (243 kDa).…”

“…This enzyme contains a highly divergent kinase catalytic domain with a fold related to conventional protein kinases (31). C-terminal to the catalytic domain, this enzyme contains a domain with a predicted ␤-propeller structure of the Kelch class (32). Notably, removal of the ␤-propeller domain was reported to cause a significant decrease in enzyme activity when assayed against the native actin-fragmin substrate (other substrates were not tested).…”

Mayven induces c-Jun expression and cyclin D1 activation in breast cancer cells

“…More recently, however, it has been suggested that the kelch repeats may also bind to proteins other than actin. For instance, Physarum actin-fragmin kinase specifically phosphorylates actin molecules that are complexed with fragmin, suggesting that this kinase is likely to recognize molecular features of both actin and fragmin (Eichinger et al, 1996). In addition, Limulus b-scruin and bovine calicin are known to be localized in a subcellular region that retains no obvious structure involving actin, implying that they may interact with different target proteins (von Bulow et al, 1995;Way et al, 1995).…”