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Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores.

Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T, Mortlock A, Keen N, Taylor SS - J. Cell Biol. (2003)

Bottom Line: Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension.Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment.Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Rd., Manchester M13 9PT, UK.

ABSTRACT
The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

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ZM447439 inhibits kinetochore localization and phosphorylation of BubR1. (A) Projections of deconvolved image stacks showing mitotic DLD-1 cells treated for 1 h with the drugs indicated and then stained to detect kinetochores/centromeres (ACA), BubR1, and DNA. (B) Bar graph plotting the fluorescence ratio of BubR1 to ACA signal under various conditions, showing that ZM447439 reduces the BubR1 signal by ∼10-fold. Values represent the mean and SEM of at least 26 different kinetochore/centromere pairs analyzed in at least three different cells. (C) Plot of BubR1 signal intensity versus interkinetochore distance at metaphase kinetochores in untreated cells (green circles) or cells exposed to ZM447439 (red diamonds) or paclitaxel (blue squares) for 40 min. ACA foci were used to determine kinetochore position. The ovals encompass at least 75% of the data points. (D) Mitotic HeLa cells collected by selective detachment after release from G1/S into the drugs indicated were analyzed by immunoblotting. In the presence of ZM447439, the phosphorylated form of BubR1 is not detectable. (E) BubR1 was affinity purified form nocodazole-arrested mitotic HeLa cells and assayed for kinase activity in the presence ZM447439. Each value represents the mean and SEM derived from three independent experiments.
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fig7: ZM447439 inhibits kinetochore localization and phosphorylation of BubR1. (A) Projections of deconvolved image stacks showing mitotic DLD-1 cells treated for 1 h with the drugs indicated and then stained to detect kinetochores/centromeres (ACA), BubR1, and DNA. (B) Bar graph plotting the fluorescence ratio of BubR1 to ACA signal under various conditions, showing that ZM447439 reduces the BubR1 signal by ∼10-fold. Values represent the mean and SEM of at least 26 different kinetochore/centromere pairs analyzed in at least three different cells. (C) Plot of BubR1 signal intensity versus interkinetochore distance at metaphase kinetochores in untreated cells (green circles) or cells exposed to ZM447439 (red diamonds) or paclitaxel (blue squares) for 40 min. ACA foci were used to determine kinetochore position. The ovals encompass at least 75% of the data points. (D) Mitotic HeLa cells collected by selective detachment after release from G1/S into the drugs indicated were analyzed by immunoblotting. In the presence of ZM447439, the phosphorylated form of BubR1 is not detectable. (E) BubR1 was affinity purified form nocodazole-arrested mitotic HeLa cells and assayed for kinase activity in the presence ZM447439. Each value represents the mean and SEM derived from three independent experiments.

Mentions: To gain insight into how ZM447439 compromises chromosome alignment and checkpoint function, we analyzed its effect on the localization of Aurora A, Aurora B, Survivin, the spindle checkpoint components BubR1 and Mad2, and the kinesin-related motor protein Cenp-E. ZM447439 did not prevent localization of Aurora A to spindle poles (unpublished data) or the localization of Aurora B and Survivin to centromeres (Fig. 6 A). However, ZM447439 did reduce kinetochore bound BubR1, Cenp-E, and Mad2 (Fig. 7 A and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200208091/DC1). Quantitation of pixel intensities shows that ZM447439 reduced kinetochore-associated BubR1 to ∼10%, both in the presence and absence of microtubule toxins (Fig. 7 B, Fig. S1, and Table SI). ZM447439 reduced kinetochore-bound Mad2 to <10% in prometaphase cells and to 30 and 43% in the presence of nocodazole and paclitaxel, respectively (Fig. S1 C). Kinetochore-bound Cenp-E was reduced to 28 and 23% in cells treated with ZM447439 and ZM447439 plus paclitaxel, respectively. However, in the presence of nocodazole and ZM447439, Cenp-E was only reduced to 59%. Interestingly, inspection of the fluorescence intensity histograms (Fig. S2) shows that in the presence of nocodazole, although ZM447439 reduced kinetochore-bound Cenp-E at the majority of kinetochores, a significant number retained normal Cenp-E levels (30% have a Cenp-E/ACA ratio greater than the mean value for kinetochores in cells treated with nocodazole only). In contrast, no kinetochores had normal BubR1 levels in the presence of ZM447439 and nocodazole (0% have a BubR1/ACA ratio greater than the mean nocodazole only value).


Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores.

Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T, Mortlock A, Keen N, Taylor SS - J. Cell Biol. (2003)

ZM447439 inhibits kinetochore localization and phosphorylation of BubR1. (A) Projections of deconvolved image stacks showing mitotic DLD-1 cells treated for 1 h with the drugs indicated and then stained to detect kinetochores/centromeres (ACA), BubR1, and DNA. (B) Bar graph plotting the fluorescence ratio of BubR1 to ACA signal under various conditions, showing that ZM447439 reduces the BubR1 signal by ∼10-fold. Values represent the mean and SEM of at least 26 different kinetochore/centromere pairs analyzed in at least three different cells. (C) Plot of BubR1 signal intensity versus interkinetochore distance at metaphase kinetochores in untreated cells (green circles) or cells exposed to ZM447439 (red diamonds) or paclitaxel (blue squares) for 40 min. ACA foci were used to determine kinetochore position. The ovals encompass at least 75% of the data points. (D) Mitotic HeLa cells collected by selective detachment after release from G1/S into the drugs indicated were analyzed by immunoblotting. In the presence of ZM447439, the phosphorylated form of BubR1 is not detectable. (E) BubR1 was affinity purified form nocodazole-arrested mitotic HeLa cells and assayed for kinase activity in the presence ZM447439. Each value represents the mean and SEM derived from three independent experiments.
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Related In: Results  -  Collection

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fig7: ZM447439 inhibits kinetochore localization and phosphorylation of BubR1. (A) Projections of deconvolved image stacks showing mitotic DLD-1 cells treated for 1 h with the drugs indicated and then stained to detect kinetochores/centromeres (ACA), BubR1, and DNA. (B) Bar graph plotting the fluorescence ratio of BubR1 to ACA signal under various conditions, showing that ZM447439 reduces the BubR1 signal by ∼10-fold. Values represent the mean and SEM of at least 26 different kinetochore/centromere pairs analyzed in at least three different cells. (C) Plot of BubR1 signal intensity versus interkinetochore distance at metaphase kinetochores in untreated cells (green circles) or cells exposed to ZM447439 (red diamonds) or paclitaxel (blue squares) for 40 min. ACA foci were used to determine kinetochore position. The ovals encompass at least 75% of the data points. (D) Mitotic HeLa cells collected by selective detachment after release from G1/S into the drugs indicated were analyzed by immunoblotting. In the presence of ZM447439, the phosphorylated form of BubR1 is not detectable. (E) BubR1 was affinity purified form nocodazole-arrested mitotic HeLa cells and assayed for kinase activity in the presence ZM447439. Each value represents the mean and SEM derived from three independent experiments.
Mentions: To gain insight into how ZM447439 compromises chromosome alignment and checkpoint function, we analyzed its effect on the localization of Aurora A, Aurora B, Survivin, the spindle checkpoint components BubR1 and Mad2, and the kinesin-related motor protein Cenp-E. ZM447439 did not prevent localization of Aurora A to spindle poles (unpublished data) or the localization of Aurora B and Survivin to centromeres (Fig. 6 A). However, ZM447439 did reduce kinetochore bound BubR1, Cenp-E, and Mad2 (Fig. 7 A and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200208091/DC1). Quantitation of pixel intensities shows that ZM447439 reduced kinetochore-associated BubR1 to ∼10%, both in the presence and absence of microtubule toxins (Fig. 7 B, Fig. S1, and Table SI). ZM447439 reduced kinetochore-bound Mad2 to <10% in prometaphase cells and to 30 and 43% in the presence of nocodazole and paclitaxel, respectively (Fig. S1 C). Kinetochore-bound Cenp-E was reduced to 28 and 23% in cells treated with ZM447439 and ZM447439 plus paclitaxel, respectively. However, in the presence of nocodazole and ZM447439, Cenp-E was only reduced to 59%. Interestingly, inspection of the fluorescence intensity histograms (Fig. S2) shows that in the presence of nocodazole, although ZM447439 reduced kinetochore-bound Cenp-E at the majority of kinetochores, a significant number retained normal Cenp-E levels (30% have a Cenp-E/ACA ratio greater than the mean value for kinetochores in cells treated with nocodazole only). In contrast, no kinetochores had normal BubR1 levels in the presence of ZM447439 and nocodazole (0% have a BubR1/ACA ratio greater than the mean nocodazole only value).

Bottom Line: Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension.Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment.Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Rd., Manchester M13 9PT, UK.

ABSTRACT
The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

Show MeSH
Related in: MedlinePlus