THE XENOPUS PROTEIN-KINASE PEG2 ASSOCIATES WITH THE CENTROSOME IN A CELL CYCLE-DEPENDENT MANNER, BINDS TO THE SPINDLE MICROTUBULES AND IS INVOLVED IN BIPOLAR MITOTIC SPINDLE ASSEMBLY

By differential screening of a Xenopus laevis egg cDNA library, we hav e isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specif ically deadenylated after fertilisation. This cDNA, called Eg2, encode s a 407 amino acid protein kinase, The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includ es Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segreg ation, respectively, A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocy te and egg extracts, as well as in lysates of Xenopus XL2 cultured cel ls. In XL2 cells, pEg2 is immunodetected only in S, G(2) and M phases of the cell cycle, where it always localises to the centrosomal region of the cell, In addition, pEg2 'invades' the microtubules at the pole s of the mitotic spindle in metaphase and anaphase, Immunoelectron mic roscopy experiments show that pEg2 is located precisely around the per icentriolar material in prophase and on the spindle microtubules in an aphase, We also demonstrate that pEg2 binds directly to taxol stabilis ed microtubules in vitro, In addition, we show that the presence of mi crotubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.