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INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization.

Xu Z, Ogawa H, Vagnarelli P, Bergmann JH, Hudson DF, Ruchaud S, Fukagawa T, Earnshaw WC, Samejima K - J. Cell Biol. (2009)

Bottom Line: Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone.This transfer requires significantly higher levels of aurora B activity.These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.

ABSTRACT
Dynamic localization of the chromosomal passenger complex (CPC) during mitosis is essential for its diverse functions. CPC targeting to centromeres involves interactions between Survivin, Borealin, and the inner centromere protein (CENP [INCENP]) N terminus. In this study, we investigate how interactions between the INCENP C terminus and aurora B set the level of kinase activity. Low levels of kinase activity, seen in INCENP-depleted cells or in cells expressing a mutant INCENP that cannot bind aurora B, are sufficient for a spindle checkpoint response when microtubules are absent but not against low dose taxol. Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone. This transfer requires significantly higher levels of aurora B activity. These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

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Active aurora B is required for aurora B and INCENP to transfer to the spindle midzone. (A–C) Localization of exogenous TrAP-INCENP (red; panels 2, 6, 10, and 14) plus endogenous aurora B (green; panels 3 and 7) or CENP-A (green; panels 11 and 15) and DNA (blue; panels 4, 8, 12, and 16) in INCENPOFF cells expressing TrAP-INCENPWT (A), TrAP-INCENPW766G (B), or TrAP-INCENPF802A (C). In all cases, TrAP-INCENP localizes to centromeres at metaphase, but in INCENPOFF cells expressing TrAP-INCENPW766G or TrAP-INCENPF802A (B and C), it fails to transfer to the spindle midzone at anaphase. Aurora B colocalizes with INCENP except for cells expressing TrAP-INCENPW766G, where it is diffuse. (D and E) INCENP and aurora B localize to centromeres in early mitosis but fail to transfer to the spindle at anaphase in wild-type clone 18 cells grown in the aurora B inhibitor ZM447439. Cells were stained for INCENP or aurora B (green; panels 3, 7, and 11) plus α-tubulin (red; panels 2, 6, and 10) and DAPI for DNA (blue; panels 4, 8, and 12). Insets show magnified views of boxed regions. Bars, 5 µm.
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fig6: Active aurora B is required for aurora B and INCENP to transfer to the spindle midzone. (A–C) Localization of exogenous TrAP-INCENP (red; panels 2, 6, 10, and 14) plus endogenous aurora B (green; panels 3 and 7) or CENP-A (green; panels 11 and 15) and DNA (blue; panels 4, 8, 12, and 16) in INCENPOFF cells expressing TrAP-INCENPWT (A), TrAP-INCENPW766G (B), or TrAP-INCENPF802A (C). In all cases, TrAP-INCENP localizes to centromeres at metaphase, but in INCENPOFF cells expressing TrAP-INCENPW766G or TrAP-INCENPF802A (B and C), it fails to transfer to the spindle midzone at anaphase. Aurora B colocalizes with INCENP except for cells expressing TrAP-INCENPW766G, where it is diffuse. (D and E) INCENP and aurora B localize to centromeres in early mitosis but fail to transfer to the spindle at anaphase in wild-type clone 18 cells grown in the aurora B inhibitor ZM447439. Cells were stained for INCENP or aurora B (green; panels 3, 7, and 11) plus α-tubulin (red; panels 2, 6, and 10) and DAPI for DNA (blue; panels 4, 8, and 12). Insets show magnified views of boxed regions. Bars, 5 µm.

Mentions: These experiments offered the first opportunity to examine the behavior of a CPC subcomplex lacking aurora B. Strikingly, although the INCENP–Survivin–Borealin subcomplex did target to centromeres, it failed to efficiently transfer to the central spindle at anaphase onset, instead remaining associated with the chromosome arms as sister chromatids separated and moved polewards (Fig. 6 B; Fig. S2, A and B; and Fig. S3, A and B). This suggests that either full CPC formation or aurora B activation is required for transfer of the complex to the spindle midzone during mitotic exit.


INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization.

Xu Z, Ogawa H, Vagnarelli P, Bergmann JH, Hudson DF, Ruchaud S, Fukagawa T, Earnshaw WC, Samejima K - J. Cell Biol. (2009)

Active aurora B is required for aurora B and INCENP to transfer to the spindle midzone. (A–C) Localization of exogenous TrAP-INCENP (red; panels 2, 6, 10, and 14) plus endogenous aurora B (green; panels 3 and 7) or CENP-A (green; panels 11 and 15) and DNA (blue; panels 4, 8, 12, and 16) in INCENPOFF cells expressing TrAP-INCENPWT (A), TrAP-INCENPW766G (B), or TrAP-INCENPF802A (C). In all cases, TrAP-INCENP localizes to centromeres at metaphase, but in INCENPOFF cells expressing TrAP-INCENPW766G or TrAP-INCENPF802A (B and C), it fails to transfer to the spindle midzone at anaphase. Aurora B colocalizes with INCENP except for cells expressing TrAP-INCENPW766G, where it is diffuse. (D and E) INCENP and aurora B localize to centromeres in early mitosis but fail to transfer to the spindle at anaphase in wild-type clone 18 cells grown in the aurora B inhibitor ZM447439. Cells were stained for INCENP or aurora B (green; panels 3, 7, and 11) plus α-tubulin (red; panels 2, 6, and 10) and DAPI for DNA (blue; panels 4, 8, and 12). Insets show magnified views of boxed regions. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig6: Active aurora B is required for aurora B and INCENP to transfer to the spindle midzone. (A–C) Localization of exogenous TrAP-INCENP (red; panels 2, 6, 10, and 14) plus endogenous aurora B (green; panels 3 and 7) or CENP-A (green; panels 11 and 15) and DNA (blue; panels 4, 8, 12, and 16) in INCENPOFF cells expressing TrAP-INCENPWT (A), TrAP-INCENPW766G (B), or TrAP-INCENPF802A (C). In all cases, TrAP-INCENP localizes to centromeres at metaphase, but in INCENPOFF cells expressing TrAP-INCENPW766G or TrAP-INCENPF802A (B and C), it fails to transfer to the spindle midzone at anaphase. Aurora B colocalizes with INCENP except for cells expressing TrAP-INCENPW766G, where it is diffuse. (D and E) INCENP and aurora B localize to centromeres in early mitosis but fail to transfer to the spindle at anaphase in wild-type clone 18 cells grown in the aurora B inhibitor ZM447439. Cells were stained for INCENP or aurora B (green; panels 3, 7, and 11) plus α-tubulin (red; panels 2, 6, and 10) and DAPI for DNA (blue; panels 4, 8, and 12). Insets show magnified views of boxed regions. Bars, 5 µm.
Mentions: These experiments offered the first opportunity to examine the behavior of a CPC subcomplex lacking aurora B. Strikingly, although the INCENP–Survivin–Borealin subcomplex did target to centromeres, it failed to efficiently transfer to the central spindle at anaphase onset, instead remaining associated with the chromosome arms as sister chromatids separated and moved polewards (Fig. 6 B; Fig. S2, A and B; and Fig. S3, A and B). This suggests that either full CPC formation or aurora B activation is required for transfer of the complex to the spindle midzone during mitotic exit.

Bottom Line: Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone.This transfer requires significantly higher levels of aurora B activity.These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.

ABSTRACT
Dynamic localization of the chromosomal passenger complex (CPC) during mitosis is essential for its diverse functions. CPC targeting to centromeres involves interactions between Survivin, Borealin, and the inner centromere protein (CENP [INCENP]) N terminus. In this study, we investigate how interactions between the INCENP C terminus and aurora B set the level of kinase activity. Low levels of kinase activity, seen in INCENP-depleted cells or in cells expressing a mutant INCENP that cannot bind aurora B, are sufficient for a spindle checkpoint response when microtubules are absent but not against low dose taxol. Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone. This transfer requires significantly higher levels of aurora B activity. These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

Show MeSH