Mutational analysis of the phosphorylation sites of the Aie1 (aurora-C) kinase in vitro

We previously reported two novel serine/threonine kinases, Aie1 (mouse) and AIE2 (human), both later referred to as aurora-C, a newly recognized member of the mitotic aurora kinase family. In the present study, we analyzed the phosphorylation sites of mouse Aiel by site-directed mutagenesis. Our results showed that protein kinase A (PKA) phosphoryIates Aiel at a threonine residue located at amino acid position 171. The T171A and T175A mutants, in which threonines located at residues 171 and 175 were replaced by alanines, revealed a significant increase in their kinase activities to phosphorylate ACS-1 (Aurora-C substrate 1). In contrast, the double mutant T171A-T175A showed impaired kinase activity. In addition, we had previously identified a PEST-like motif located at the N terminus of Aiel. Mutation analysis in the present study revealed that the quadruple mutant in which the PEST-like motif was mutated significantly abrogated Aiel kinase activity. This is the first report of the analysis of potential phosphorylation sites of mouse aurora-C in vitro.