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The non-catalytic domain of the Xenopus laevis auroraA kinase localises the protein to the centrosome

Régis Giet^* and Claude Prigent

Groupe Cycle Cellulaire, UMR 6061 Génétique et Développement, CNRS – Université de Rennes I, IFR 97 Génomique Fonctionnelle et Santé, Faculté de Médecine, 2 avenue du Pr Léon Bernard, CS 34317, 35043 Rennes cedex, France
^* Present address: University of Cambridge, Department of Genetics, Downing Street, Cambridge, CB2 3EH, UK

View larger version (60K): [in a new window]   Fig. 1. Sequence comparison of vertebrate auroraA kinase non-catalytic domains, recombinant Xenopus auroraA N-terminal protein (Nt-pEg2-(His)6) and antibody specificity. (A) Sequence alignment of vertebrate auroraA N-terminal domains. Xl: Xenopus laevis pEg2, GenBank accession no. Z177207 (Roghi et al., 1998); Mm: Mus musculus AIR1, GenBank accession no. U69106) (Shindo et al., 1998); Hs: Homo sapiens aurora2, GenBank accession no. AF008551 (Shindo et al., 1998). Identical amino acids are shown in grey. (B) Nt-pEg2-(His)6 protein was overexpressed in E. coli and purified by affinity chromatography on a Ni-NTA agarose column. The protein was eluted with 250 mM imidazole and concentrated through a centricon 10. 2 µl (8 µg) were analysed on a 20% SDS-polyacrylamide gel stained with Coomassie Blue. (C) Specificity of 1C1 and 6E3 monoclonal antibodies. 1 µl of a Xenopus egg extract containing about 40 ng of endogenous pEg2 (lanes 2 and 4) or 40 ng of Nt-pEg2-(His)6 purified protein (lanes 1 and 3) were subjected to electrophoresis on a 20% SDS-polyacrylamide gel, transferred on nitrocellulose membrane and probed with 1C1 (lanes 1 and 2) or 6E3 (lanes 3 and 4) monoclonal antibodies (dilution 1/100). The positions of molecular mass (kDa) markers in B and C are shown.

View larger version (33K): [in a new window]   Fig. 2. The N-terminal domain of pEg2 does not affect the activity of the full-length kinase and does not bind to XlEg5. (A) The pEg2 kinase activity of the recombinant pEg2-(His)6 protein was assayed in vitro using myelin basic protein (MBP) as a substrate in the presence of increasing amounts of either the recombinant inactive pEg2-K/R-(His)6 kinase or the recombinant N-terminal domain Nt-pEg2-(His)6 protein. After incubation in the presence of [-32P] ATP, the reaction mixture was subjected to SDS-polyacrylamide gel electrophoresis and electrotransferred onto a nitrocellulose membrane. The radioactive MBP was counted with a Phosphoimager. The kinase activity is expressed as a percentage of the activity without inhibitor. The kinase activity was estimated in the presence of the inactive kinase (filled circles) or the N-terminal domain of pEg2 (open circles). (B) Affinity chromatography. A Ni-NTA agarose column saturated with either Fl-pEg2-(His)6 (full length), Nt-pEg2-(His)6 (N-terminal) or no protein (control) was loaded with 200 µl of Xenopus CSF extract. Affinity-bound proteins in the different columns were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. The membranes were cut and the upper part (>70 kDa) was incubated with anti-XlEg5 polyclonal antibodies (dilution 1/1000), while the lower part (<70 kDa) was incubated with the anti-pEg2 1C1 mAb (diluted 1/100). Lane 1, Xenopus egg extracts; lanes 2-5, elution fractions; Fl-pEg2-(His)6 column, nickel column preloaded with the full-length pEg2-(His)6 recombinant protein; Nt-pEg2-(His)6 column, nickel column preloaded with pEg2 N-terminal-(His)6 recombinant protein; Control column, control nickel column without any recombinant protein.

View larger version (59K): [in a new window]   Fig. 3. The N-terminal domain of pEg2 shows much less affinity than the full-length protein for paclitaxel-stabilised microtubules in vitro. Microtubules were polymerised in vitro in the presence of purified bovine brain tubulin (lanes 3 and 4), stabilised with paclitaxel and centrifuged through a glycerol cushion to separate microtubules and microtubule associated protein (pellet, P) from proteins that do not associate to microtubules (supernatant, S). In a control reaction, purified tubulin was replaced by bovine serum albumin (lanes 1 and 2). In the presence of paclitaxel, the ß-tubulin is recovered in the pellet (A and C, lane 4) whereas the bovine serum albumin remains in the supernatant (A and C, lane 1). Purified recombinant pEg2-(His)6 (A and B) or purified recombinant Nt-pEg2-(His)6 (C and D) were also incorporated to the reaction. The pellet (P) and the supernatant (S) were analysed for the presence of recombinant proteins using western blotting with the 1C1 monoclonal antibody (diluted 1:100).

View larger version (58K): [in a new window]   Fig. 4. The N-terminal domain of pEg2 binds to centrosomes assembled in Xenopus egg extract from sperm heads. Centrosomes were assembled in vitro by incubating demembranated sperm heads in Xenopus egg CSF extract (Stearns and Kirschner, 1994) in the absence (A-H) or presence (I-P) of 400 ng/µl of the recombinant Nt-pEg2-(His)6 protein. The reaction was performed in the absence (A-D and I-L) or presence of the microtubule depolymerising drug nocodazole (20 µM final concentration) (E-H and M-P). (A,E,I,M) Hoechst-stained DNA. (C,G) The endogenous pEg2 protein probed with mouse 1C1 monoclonal antibody (diluted 1:50). (K,O) The recombinant Nt-pEg2-(His)6 protein probed with mouse 6E3 monoclonal antibody (diluted 1:50). (B,F,JN) Centrosome stained with -tubulin antibody (diluted 1:1000). Fluorescein-conjugated anti-rabbit antibodies (diluted 1:500) and Texas Red-conjugated anti-mouse antibodies (diluted 1:500) were used as secondary antibodies. (D,H,L,P) Overlay. Scale bar, 10 µm. The localisation of the proteins was observed by fluorescence microscopy (DMRXA Leica); the images were acquired using a black and white camera and treated with the Leica-Q-Fish program.

View larger version (34K): [in a new window]   Fig. 5. The N-terminal domain of pEg2 fused to GFP localises to the centrosome in XL2 cells. GFP, Nt-pEg2-GFP and Cd-pEg2-GFP proteins were constitutively expressed in Xenopus XL-2 cells using transient transfection with the pEGFPN1 expression vector. Cells were fixed on coverslips and processed for immunofluorescence. (A-D) GFP transfected cells (control), (E-H) Nt-pEg2-GFP transfected cells, (I-L) Dc-pEg2-GFP transfected cells. (A,E,I) Hoechst-stained DNA. (B,F,J) -tubulin staining with a rabbit polyclonal antibody (diluted 1:1000), revealed with a Texas Red-conjugated secondary antibody (diluted 1:500). (C,G,K) The localisation of the GFP proteins. (D,H,L) Overlay. Scale bar, 10 µm. The localisation of the proteins was observed by fluorescence microscopy (DMRXA Leica); the images were acquired using a black and white camera and treated with the Leica-Q-Fish program

View larger version (16K): [in a new window]   Fig. 6. The localisation of the pEg2 N terminus is microtubule-dependent. Xenopus XL2 cells expressing Nt-pEg2-GFP or Cd-pEg2-GFP protein were treated for 6 hours with the microtubule depolymerising drug nocodazole (10 µg/ml). The number of cells showing a centrosome localisation of the GFP fusion protein was then estimated. White bars, cells observed without nocodazole; black bars, cells after nocodazole treatment. Values are means ± s.e.m.

View larger version (40K): [in a new window]   Fig. 7. The N-terminal domain of pEg2 inhibits bipolar spindle assembly and destabilises previously assembled bipolar spindles in Xenopus egg extract. The bipolar mitotic spindle assembly assay was performed as previously described (Roghi et al., 1998). After fixation, the spindles were scored under a fluorescence microscope. (A) Bipolar spindle (top) and monopolar spindle (bottom). The spindle incorporates rhodamine-labelled tubulin and appears in red. DNA is stained by Hoechst dye (blue). (B) 400 ng/µl (final concentration) of either the Nt-pEg2-(His)6 protein or a control pMAL peptide were added during spindle assembly. In the presence of Nt-pEg2-(His)6, only 34±18% (3 different experiments) of the spindles remained bipolar instead of 81±12% (4 different experiments) in the control. (C) Bipolar spindles previously assembled in Xenopus egg extract were incubated for 1 hour with 400 ng/µl of Nt-pEg2(His)6 protein, or with the control pMAL peptide at the same concentration. In the presence of Nt-pEg2-(His)6, 55±5% (3 different experiments) of the spindles remained bipolar instead of 82±8% in the control (4 different experiments).

View larger version (26K): [in a new window]   Fig. 8. Localisation mechanism of pEg2. The N-terminal domain (red) localises the kinase to the centrosomes (grey) through an interaction with a hypothetical protein (blue) that binds to microtubules (black), the localisation being microtubule-dependent. Then the kinase binds to its substrates (yellow), which stabilises the localisation so that it becomes microtubule-independent.