Roghi C et al. (1998), The Xenopus protein kinase pEg2 associates with...

XB-ART-14958

J Cell Sci 1998 Mar 01;111 ( Pt 5):557-72. doi: 10.1242/jcs.111.5.557.

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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.

Roghi C , Giet R , Uzbekov R , Morin N , Chartrain I , Le Guellec R , Couturier A , Dorée M , Philippe M , Prigent C .

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By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 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 poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules 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.

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Species referenced: Xenopus laevis
Genes referenced: aurka aurkb

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	Fig. 5. Immuno-localisation of pEg2 during the cell cycle. The monoclonal anti-pEg2 antibody (1C1) was used to immunolocalise pEg2 in Xenopus XL2 cells. Cells were grown and fixed as described in Materials and Methods and labeled for indirect immuofluorescence microscopy with various antibodies: anti-g-tubulin rabbit polyclonal antibody (B,F,J,N), anti-pEg2 1C1 monoclonal antibody (C,G,K,O), anti-pEg2 1C1 monoclonal and anti-g-tubulin rabbit polyclonal antibodies (D,H,L,P). In several places, co-distribution of red fluorescence from pEg2 and green fluorescence from g-tubulin labelling was significant enough to produce a yellow signal, indicating an overlap in the distribution of the two proteins. Cells were also observed using phase contrast microscopy (A,E,I,M) to determine the stage of the cell cycle. The cell in A to D is in G1/early S phase, E to H is in late S phase/G2, I to L is in prophase and M to P is in metaphase. Bar, 10 mm.
	Fig. 6. Immunolocalisation of pEg2 at different mitotic stages. The monoclonal anti-pEg2 antibody (1C1) was used to immunolocalise pEg2 in Xenopus XL2 cells at different stages of mitosis. Cells were observed using phase contrast microscopy (A,C,E,G) to determine the stage of mitosis. Cell in A and B is in prophase; in C and D in prometaphase; in E and F in anaphase and in G and H in telophase Cells were labelled for indirect immunofluorescence microscopy using anti-pEg2 1C1 monoclonal antibody (B,D,F,H). Bar, 10 mm.
	Fig. 7. Ultrastructural localisation of pEg2 in mitotic cells. Xenopus XL2 cells were grown on coverslips, fixed in cold methanol (-20), incubated with the monoclonal antibody 1C1 and processed for electron microscopy as described in Materials and Methods. pEg2 has been detected using the 1C1 monoclonal antibody and revealed with a secondary antibody coupled to 15 nm gold particles. (A) A centrosome in prophase, (B) enlargement of the right pole of the spindle in metaphase (the metaphase plate is shown in the panel in the bottom right corner) and (C) one pole of a spindle in anaphase (the position of chromosomes is shown in the lower panel). Bars: 1 mm (A, B and C); 0.5 mm (insets in B and C).