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 Aie1 by site-directed mutagenesis. Our results showed that protein kinase A (PKA) phosphorylates Aie1 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 Aie1. Mutation analysis in the present study revealed that the quadruple mutant in which the PEST-like motif was mutated significantly abrogated Aie1 kinase activity. This is the first report of the analysis of potential phosphorylation sites of mouse aurora-C in vitro.
Chen
Sheau-Hu
SH
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC.
Tang
Tang K
TK
D000818:Animals; D064108:Aurora Kinase C; D064127:Aurora Kinases; D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D066298:In Vitro Techniques; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D017931:DNA Primers; D010446:Peptide Fragments; D013912:Threonine; C576927:AURKC protein, human; C576928:Aurkc protein, mouse; D064108:Aurora Kinase C; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases