Abstract
We have previously reported on the cloning of XlEg5, a Xenopus laevis kinesin-related protein from the bimC family (Le Guellec, R., Paris, J., Couturier, 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., Dorée, 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 Xenopus XL2 cells, pEg2 and XlEg5 are both confined to separated centrosomes in prophase, and then to the microtubule spindle poles. We also show that pEg2 co-immunoprecipitates with XlEg5 from egg extracts and XL2 cell lysates. Both proteins can directly interact in vitro, but also through the two-hybrid system. Furthermore immunoprecipitated pEg2 were found to remain active when bound to the beads and phosphorylate XlEg5 present in the precipitate. Two-dimensional mapping of XlEg5 tryptic peptides phosphorylated in vivo first confirmed that XlEg5 was phosphorylated by p34(cdc2) and next revealed that in vitro pEg2 kinase phosphorylated XlEg5 on the same stalk domain serine 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.
Highlights
Mitotic spindle assembly is a complex and essential event in the cell division process because it is a prerequisite for chromosome segregation [1]
We have previously reported on the cloning of XlEg5, a Xenopus laevis kinesin-related protein from the bimC family (Le Guellec, R., Paris, J., Couturier, A., Roghi, C., and Philippe, M. (1991) Mol
Inhibition of either XlEg5 or pEg2 activity during mitosis in Xenopus egg extract led to monopolar spindle formation
Summary
XlEg5, X. laevis kinesin-like protein [9], XKCM1 [10]), cytoplasmic dynein [11], dynactin [12], NuMA [13], protein kinases like p34cdc2 [3], Plx [14, 15], pEg2 [16], and protein phosphatases PP1 and PP2A [17] Motor protein activities such as association with microtubules, interaction with other proteins, and subcellular localization are regulated by phosphorylation reactions (18 –21). In Xenopus egg extracts, both the inhibition of XlEg5 through the addition of antibodies [34] and the inhibition of pEg2 through the addition of an inactive dominant negative pEg2 form [16] provoke an inhibition of the mitotic spindle assembly These results suggest that pEg2 may act on a substrate involved in centrosome separation, and one of the obvious candidates is the kinesinrelated protein XlEg5. Our results definitely suggest that XlEg5 is phosphorylated in vivo on two residues with two different kinases; a threonine residue is phosphorylated by p34cdc, and a serine is phosphorylated by pEg2
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