The Xenopus laevis aurora-related protein kinase pEg2 associates with and phosphorylates the kinesin-related protein XlEg5

We have previously reported on the cloning of XlEg5, a Xenopus laevis kines in-related protein from the bimC family (Le Guellec, R., Paris, J., Couturi er, A., Roghi, C., and Philippe, M. (1991) Mol. Cell. Biol. 11, 3395-3408) as well as pEg2, an Aurora-related serine/threonine kinase (Roghi, C., Giet , R., Uzbekov, R., Morin, N., Chartrain, I., Le Guellec, R., Couturier, A., Doree, M., Philippe, M., and Prigent, C. (1998) J. Cell Sci. 111, 557-572) . Inhibition of either XlEg5 or pEg2 activity during mitosis in Xenopus egg extract led to monopolar spindle formation. Here, we report that in Xenopu s XL2 cells, pEg2 and XlEg5 are both confined to separated centrosomes in p rophase, and then to the microtubule spindle poles. We also show that pEg2 co-immunoprecipitates with XlEg5 from egg extracts and XL2 cell lysates. Bo th proteins can directly interact in vitro, but also through the two-hybrid system. Furthermore immunoprecipitated pEg2 were found to remain active wh en bound to the beads and phosphorylate XlEg5 present in the precipitate. T wo-dimensional mapping of XlEg5 tryptic peptides phosphorylated in vivo fir st confirmed that XlEg5 was phosphorylated by p34(cdc2) and next revealed t hat in vitro pEg2 kinase phosphorylated XlEg5 on the same stalk domain seri ne residue that was phosphorylated in metabolically labeled XL2 cells. The kinesin-related XlEg5 is to our knowledge the first in vivo substrate ever reported for an Aurora-related kinase.