Exploring the functional interactions between Aurora B, INCENP, and survivin in mitosis

Abstract

The function of the Aurora B kinase at centromeres and the central spindle is crucial for chromosome segregation and cytokinesis, respectively. Herein, we have investigated the regulation of human Aurora B by its complex partners inner centromere protein (INCENP) and survivin. We found that overexpression of a catalytically inactive, dominant-negative mutant of Aurora B impaired the localization of the entire Aurora B/INCENP/survivin complex to centromeres and the central spindle and severely disturbed mitotic progression. Similar results were also observed after depletion, by RNA interference, of either Aurora B, INCENP, or survivin. These data suggest that Aurora B kinase activity and the formation of the Aurora B/INCENP/survivin complex both contribute to its proper localization. Using recombinant proteins, we found that Aurora B kinase activity was stimulated by INCENP and that the C-terminal region of INCENP was sufficient for activation. Under identical assay conditions, survivin did not detectably influence kinase activity. Human INCENP was a substrate of Aurora B and mass spectrometry identified three consecutive residues (threonine 893, serine 894, and serine 895) containing at least two phosphorylation sites. A nonphosphorylatable mutant (TSS893-895AAA) was a poor activator of Aurora B, demonstrating that INCENP phosphorylation is important for kinase activation.

Figures

Figure 11.

Ala mutants of INCENP C3 fragment are less effective in Aurora B activation. (A) The GST-INCENP mutants C7, C3, C3(3A), or C3(4A) and His-Aurora B were coexpressed in Sf-9 cells and the complexes purified as described in the legend to Figure 9. INCENP and Aurora B protein levels were determined by Western blotting. (B) Kinase activities associated with the samples described in A were measured with or without exogenous histone H3; the bottom panel shows a prolonged exposure to illustrate phosphorylation of GST-INCENP fragments. (C) GST-Aurora B and INCENP C3 (WT or 3A) were purified separately, and after mixing of the proteins, phosphorylation of INCENP was assayed (top); the presence of equal amounts of INCENP substrate was demonstrated by Coomassie Blue staining (CBB; bottom). (D) The effect of adding increasing amounts of INCENP C3 (WT or 3A) to Aurora B was measured by in vitro kinase assays, with histone H3 (top) or MBP (bottom) as substrates.

Figure 1.

A catalytically inactive Aurora B acts as a dominant-negative mutant. (A) Inducible T-Rex U2OS cell lines (Invitrogen) harboring myc-tagged K106R mutant or wild-type (wt) human Aurora B transgenes were treated with or without tetracycline (1 μg/ml final concentration). After 8 h, nocodazole (or dimethyl sulfoxide) was added and cells were incubated for an additional 16 h. Myc-tagged Aurora B was immunoprecipitated with anti-myc antibody, and protein amounts and kinase activities in immunoprecipitates were determined by Western blotting (top) and in vitro kinase assays (bottom). (B) Myc-tagged K106R or wild-type Aurora B were immunoprecipitated with anti-myc antibody from lysates used in A. Immunoprecipitates were subjected to Western blotting with antibodies against myc-Aurora B and INCENP; whole-cell lysates were analyzed in parallel. (C) K106R and wild-type Aurora B expressing cells were treated for 24 h with or without tetracycline and then analyzed by immunofluorescence microscopy with anti-myc antibodies. DNA was counterstained with DAPI. (D) Histogram shows a quantitative analysis of the extent of multinucleation at different times of wild-type or K106R mutant Aurora B expression.

Figure 2.

Mislocalization of the K106R Aurora B mutant in mitosis. T-Rex U2OS cell lines expressing myc-tagged Aurora B wild-type and K106R mutant were fixed 24 h after induction and subjected to immunofluorescence microscopy, by using the anti-myc antibody. DNA was counterstained with DAPI. (A) prometaphase (B) anaphase and telophase stages. Red, myc; blue, DAPI.

Figure 3.

Localization of INCENP and survivin in cells expressing the K106R mutant of Aurora B. T-Rex U2OS cell lines expressing myc-tagged K106R mutant Aurora B were incubated for 10 h in the presence or absence of tetracycline, fixed and then stained with antibodies against INCENP, survivin, or CENP-B (green). DNA was counterstained with DAPI (blue). Prometaphase stage cells are shown.

Figure 4.

Depletion of INCENP or survivin by siRNA results in mitotic defects and multinucleation. (A) Aurora B and INCENP protein levels in asynchronously growing (lane 1) or nocodazole treated (lanes 2 and 3) HeLa S3 cell extracts. To detect the Aurora B protein by Western blotting, two distinct antibodies were used, one directed against the C terminus (lanes 1 and 2), the other against the N terminus (lane 3). (B) Aurora B, INCENP, surviving, and α-tubulin levels after 48 h treatment with siRNA directed to Aurora B, INCENP, or survivin, as indicated. The GL-2 duplex was used for control. (C) Depletion of INCENP or survivin caused multinucleation. U2OS cells were fixed 48 h after siRNA treatment and stained with anti-α-tubulin antibodies (green). DNA was counterstained with DAPI (blue). (D) Mitotic defects in INCENP- or survivin-depleted cells, 48 h after treatment with siRNA. α-Tubulin staining: red; DNA staining: blue.

Figure 5.

Lack of centromere localization of Aurora B/INCENP/survivin after depletion of individual proteins by siRNA. After treatment of T-Rex-U2OS cells for 48 h by siRNA duplexes directed against Aurora B, INCENP, or survivin, all three proteins were localized by immunofluorescence microscopy. DNA was counterstained with DAPI.

Figure 6.

Cell cycle analysis of Aurora B, INCENP, and survivin. (A) Aurora B, INCENP, survivin, cyclin B, and α-tubulin protein levels through the cell cycle were determined by Western blotting. HeLa S3 cells were synchronized by an arrest-release protocol from an aphidicolin/thymidine block at the G1/S boundary (time 0). For comparison, cells were also analyzed after a nocodazole induced prometaphase arrest (noc). Arrows point to the full-length, wild-type Aurora B, whereas the arrowhead and asterisk denote the slower migrating form of INCENP. (B) Aurora B kinase activity at the corresponding cell cycle stages was measured in immunoprecipitates, by using histone H3 as a substrate, and analyzed using a PhosphorImager (bottom). Amounts of immunoprecipitated Aurora B protein were determined by Western blotting (top). A sample of the nocodazole-treated cell lysate before immunoprecipitation [lys(noc)] was analyzed in parallel. The numbers indicate the relative specific activity of Aurora B, with the value at the 4-h time point arbitrarily set to 1.0.

Figure 7.

Effects of INCENP and survivin on Aurora B kinase activity. GST-Aurora B, GST-Aurora B/His-survivin, GST-Aurora B/His-INCENP, and GST-Aurora B/His-survivin/His-INCENP complexes were coexpressed in Sf-9 cells and purified using glutathione-Sepharose. Aurora B protein levels were determined by Western blotting, and kinase activity was measured in in vitro kinase assays, with or without MBP as an exogenous substrate. Phosphate incorporation was analyzed using a PhosphorImager. INCENP and survivin copurifying with Aurora B were also detected by Western blotting. MBP was visualized by Coomassie Blue staining (CBB).

Figure 8.

Phosphorylation of INCENP by Aurora B. GST-Aurora B and GST-Aurora B/His-INCENP complexes were purified using glutathione-Sepharose, and Aurora B protein levels were determined by Western blotting (left). The complexes were then used for in vitro kinase reactions, with or without histone H3 as an exogenous substrate. Phosphate incorporation was detected using a PhosphorImager (right, top) and histone H3 visualized by CBB (bottom). Note the incorporation of phosphate into both INCENP and Aurora B (arrows).

Figure 9.

Mapping of INCENP domains required for activation of Aurora B. (A) Schematic representation of INCENP deletion mutants (C1–C8). The amino acid sequences of deletion mutants C3–C8 are indicated using the single-letter code. The regions of INCENP involved in Aurora B binding (822–877) and activation (878–892), as well as the domain serving as a substrate for phosphorylation (893–919), were deduced from the data shown in B. (B) The GST-INCENP deletion mutants (C3–C8) were coexpressed with His-Aurora B in Sf-9 cells and isolated on glutathione-Sepharose beads. The GST-INCENP mutants and coprecipitating His-Aurora B were then detected by Western blotting with anti-GST and anti-Aurora B antibodies, respectively. Phosphorylation of the INCENP mutants and histone H3 kinase activity of all samples were tested in parallel.

Figure 10.

Identification of major phosphorylation sites within INCENP by mass spectrometry. Matrix-assisted laser desorption ionization/time of flight mass spectrum of a tryptic digest of the INCENP C2 fragment (785–919) phosphorylated by Aurora B in vitro. A selected mass range containing the unphosphorylated (0 ph), singly (1 ph), doubly (2 ph), and triply phosphorylated (3 ph) peptide (893–907) TSSAVWNSPPLQGAR is shown. The identity and phosphorylation states of these peptides were confirmed using postsource decay analysis and electrospray tandem mass spectrometry (our unpublished data) as described in MATERIALS AND METHODS. The spectrum was recorded in negative ion reflector mode using 2,5 dihydroxybenzoic acid as matrix.

Figure 12.

INCENP phosphorylation and Aurora B activation. (A) Full-length INCENP with mutated TSS motif barely activates Aurora B. Complexes of Aurora B and either wild-type or 3A mutant full-length INCENP were formed in Sf-9 cells and purified using glutathione-Sepharose beads. Recovery of proteins was determined by Western blotting (top and middle) and kinase activity of the complexes measured using histone H3 as a substrate (bottom). Lane 1, GST-INCENP (3A)/His-Aurora B; lane 2, GST-INCENP (WT)/His-Aurora B; lane 3, His-INCENP (3A)/GST-Aurora B; and lane 4, His-INCENP (WT)/GST-Aurora B. (B) Residual phosphorylation of INCENP with mutated TSS motif. Phosphate incorporation into INCENP was tested by subjecting Aurora B/INCENP complexes to in vitro kinase reactions. Protein levels of Aurora B and INCENP were determined by Western blotting, and the kinase activity of each complex was measured with histone H3 as substrates. Lane 1, GST-Aurora B(WT)/His-INCENP(WT); lane 2, GST-Aurora B(WT)/His-INCENP(3A); and lane 3, GST-Aurora B(K106R)/His-INCENP(3A).

Figure 13.

Aurora B phosphorylation sites in INCENP. The C termini of INCENP proteins of all listed species, except for budding yeast, show a highly conserved phosphorylation motif. This motif conforms to an emerging consensus motif for Aurora B phosphorylation sites (see text).