Despite the central importance of the mitotic spindle in cell division, the regulation of mitotic spindle formation and function is not well understood. Our lab has previously identified a C. elegans orthologue of the Aurora kinase family, AIR-2, that is required for proper chromosome segregation and cytokinesis. AIR-2 localizes to chromosomes at metaphase, the central spindle at anaphase, and to the cytokinesis remnant at telophase.(1) This dynamic localization pattern is shared by a group of proteins known as chromosomal passengers, which includes the C. elegans inner centromeric protein ICP-1. ICP-1 co-localizes with AIR-2, and may act to target AIR-2 as it is required to localize AIR-2 in vivo, and directly binds AIR-2 in vitro.(2) Furthermore,
icp-1(RNAi) results in a phenotype that is indistinguishable from
air-2(RNAi).( 2) Given these results, we hypothesized that ICP-1 is a direct substrate of AIR-2. Here we show that recombinant AIR-2 specifically phosphorylates recombinant ICP-1 in vitro. To localize the site of AIR-2 phosphorylation in ICP-1, constructs encoding the amino-terminus (IN), middle (IM), and carboxy-terminus (IC) of ICP-1 were created. In vitro kinase assays revealed that only the IC fragment could be phosphorylated by AIR-2. Site-direct mutagenesis of two adjacent serines in the IC fragment that are highly conserved across eukaryotes strongly reduced the level of phosphorylation by AIR-2. Phosphorylation of these serines was additive as mutating each residue separately caused only partial reduction in phosphorylation. Interestingly, both full length ICP-1 and the IC fragment, but not the IN or IM fragments, induce an increase in the kinase activity of AIR-2 towards the generic substrate MBP. This induction requires that ICP-1 be phosphorylated by AIR-2 as mutation of both of the serines at the AIR-2 phosphorylation site in either full-length ICP-1 or the IC fragment abolished the ability to increase AIR-2 activity. In conclusion, ICP-1 is a direct substrate of AIR-2 kinase, and phosphorylation of ICP-1 feeds back to potentiate the kinase activity of AIR-2. We are currently creating transgenic lines that express GFP fusions of either wildtype or phosphorylation-site mutant ICP-1 to determine the role of AIR-2 mediated phosphorylation in ICP-1 localization and function in vivo. (1) Schumacher et al., (1998) Journal of Cell Biology143:1635-1646. (2) Kaitna et al.,(2000) Current Biology10:1172-1181.