Limits...
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.

Show MeSH
Quantitation of aurora B kinase activity in INCENP mutants. (A) Estimation of aurora B activity by immunoblotting. Asynchronous cells were harvested after treatment with doxycycline for 28 h or with 2 µM ZM447439 for 5 h (Fig. S1 C), and lysates were subjected to immunoblotting with the indicated antibodies. α-Tubulin and haspin kinase substrate H3T3ph are shown as controls. White lines indicate that intervening lanes have been spliced out. (B) Measurement of H3S10ph levels in the same samples using Odyssey. (C) Ratio of aurora B protein levels versus the loading control α-tubulin for each sample as measured using Odyssey. (D) Mitotic index of each cell line at the time of harvesting. (E) Diagram showing the relative levels of aurora B activity based on the level of H3S10ph measured using the Odyssey assay. WT, wild type. Error bars indicate SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2806593&req=5

fig5: Quantitation of aurora B kinase activity in INCENP mutants. (A) Estimation of aurora B activity by immunoblotting. Asynchronous cells were harvested after treatment with doxycycline for 28 h or with 2 µM ZM447439 for 5 h (Fig. S1 C), and lysates were subjected to immunoblotting with the indicated antibodies. α-Tubulin and haspin kinase substrate H3T3ph are shown as controls. White lines indicate that intervening lanes have been spliced out. (B) Measurement of H3S10ph levels in the same samples using Odyssey. (C) Ratio of aurora B protein levels versus the loading control α-tubulin for each sample as measured using Odyssey. (D) Mitotic index of each cell line at the time of harvesting. (E) Diagram showing the relative levels of aurora B activity based on the level of H3S10ph measured using the Odyssey assay. WT, wild type. Error bars indicate SD.

Mentions: Aurora B kinase activity was significantly decreased in INCENPOFF/TrAP-INCENPW766G cells. As measured by quantitative immunoblotting, levels of H3S10ph fell essentially to those seen in INCENPOFF cells (Fig. 5, A, B, and E). A similar decline in the levels of H3S10ph and H3S28ph was observed by indirect immunofluorescence (Fig. S3 F). These data support the accepted notion that INCENP–aurora B complex formation is required for full kinase activation (Adams et al., 2000; Bishop and Schumacher, 2002; Bolton et al., 2002; Honda et al., 2003).


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)

Quantitation of aurora B kinase activity in INCENP mutants. (A) Estimation of aurora B activity by immunoblotting. Asynchronous cells were harvested after treatment with doxycycline for 28 h or with 2 µM ZM447439 for 5 h (Fig. S1 C), and lysates were subjected to immunoblotting with the indicated antibodies. α-Tubulin and haspin kinase substrate H3T3ph are shown as controls. White lines indicate that intervening lanes have been spliced out. (B) Measurement of H3S10ph levels in the same samples using Odyssey. (C) Ratio of aurora B protein levels versus the loading control α-tubulin for each sample as measured using Odyssey. (D) Mitotic index of each cell line at the time of harvesting. (E) Diagram showing the relative levels of aurora B activity based on the level of H3S10ph measured using the Odyssey assay. WT, wild type. Error bars indicate SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806593&req=5

fig5: Quantitation of aurora B kinase activity in INCENP mutants. (A) Estimation of aurora B activity by immunoblotting. Asynchronous cells were harvested after treatment with doxycycline for 28 h or with 2 µM ZM447439 for 5 h (Fig. S1 C), and lysates were subjected to immunoblotting with the indicated antibodies. α-Tubulin and haspin kinase substrate H3T3ph are shown as controls. White lines indicate that intervening lanes have been spliced out. (B) Measurement of H3S10ph levels in the same samples using Odyssey. (C) Ratio of aurora B protein levels versus the loading control α-tubulin for each sample as measured using Odyssey. (D) Mitotic index of each cell line at the time of harvesting. (E) Diagram showing the relative levels of aurora B activity based on the level of H3S10ph measured using the Odyssey assay. WT, wild type. Error bars indicate SD.
Mentions: Aurora B kinase activity was significantly decreased in INCENPOFF/TrAP-INCENPW766G cells. As measured by quantitative immunoblotting, levels of H3S10ph fell essentially to those seen in INCENPOFF cells (Fig. 5, A, B, and E). A similar decline in the levels of H3S10ph and H3S28ph was observed by indirect immunofluorescence (Fig. S3 F). These data support the accepted notion that INCENP–aurora B complex formation is required for full kinase activation (Adams et al., 2000; Bishop and Schumacher, 2002; Bolton et al., 2002; Honda et al., 2003).

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