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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 Aurora A and Aurora B. (A) Chemical structure of ZM447439. (B) Table showing the IC50 values (μM) of ZM447439 against a panel of protein kinases. (C) DNA content histograms of HeLa, A549, and HME cells treated with ZM447439 for the times indicated in hours. (D) Mitotic DLD-1 cells stained for phosphohistone H3 (green) and DNA (red).
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fig1: ZM447439 inhibits Aurora A and Aurora B. (A) Chemical structure of ZM447439. (B) Table showing the IC50 values (μM) of ZM447439 against a panel of protein kinases. (C) DNA content histograms of HeLa, A549, and HME cells treated with ZM447439 for the times indicated in hours. (D) Mitotic DLD-1 cells stained for phosphohistone H3 (green) and DNA (red).

Mentions: To identify novel Aurora inhibitors, ∼250,000 compounds were screened for the ability to inhibit the kinase activity of recombinant human Aurora A against an artificial peptide substrate. One inhibitor identified was further modified to produce ZM447439 (4-(4-(N-benzoylamino)anilino)-6-methoxy-7-(3-(1-morpholino)propoxy)quinazoline; Fig. 1 A). In in vitro kinase assays using purified recombinant proteins, ZM447439 inhibited Aurora A and B with IC50 values of 110 and 130 nM, respectively (Fig. 1 B). In contrast, the majority of other protein kinases assayed were not inhibited by ZM447439. Based on data from model systems, we predicted that an Aurora inhibitor should prevent cell division and inhibit phosphorylation of histone H3 on serine 10. To determine whether ZM447439 inhibits cell division, a panel of human cell lines was treated with 2 μM ZM447439 for up to 96 h (Fig. 1 C). After 18 h, the vast majority of cells in all the lines had 4N DNA contents. All the lines analyzed then endoreduplicated, accumulating cells with DNA contents greater than 4N, demonstrating that ZM447439 completely inhibits cell division. To determine whether ZM447439 inhibits phosphorylation of histone H3 on serine 10, untreated and ZM447439-treated cells were stained with an anti-phosphohistone H3 antibody (Hsu et al., 2000). In untreated cells, chromosomes stained positive for phosphohistone H3 (Fig. 1 D). However, after brief exposures to ZM447439, phosphohistone H3 was not detectable, demonstrating that ZM447439 does inhibit mitotic phosphorylation of histone H3. Consistent with previous observations (Adams et al., 2001c), the lack of histone H3 phosphorylation did not appear to affect chromosome condensation.


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 Aurora A and Aurora B. (A) Chemical structure of ZM447439. (B) Table showing the IC50 values (μM) of ZM447439 against a panel of protein kinases. (C) DNA content histograms of HeLa, A549, and HME cells treated with ZM447439 for the times indicated in hours. (D) Mitotic DLD-1 cells stained for phosphohistone H3 (green) and DNA (red).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172902&req=5

fig1: ZM447439 inhibits Aurora A and Aurora B. (A) Chemical structure of ZM447439. (B) Table showing the IC50 values (μM) of ZM447439 against a panel of protein kinases. (C) DNA content histograms of HeLa, A549, and HME cells treated with ZM447439 for the times indicated in hours. (D) Mitotic DLD-1 cells stained for phosphohistone H3 (green) and DNA (red).
Mentions: To identify novel Aurora inhibitors, ∼250,000 compounds were screened for the ability to inhibit the kinase activity of recombinant human Aurora A against an artificial peptide substrate. One inhibitor identified was further modified to produce ZM447439 (4-(4-(N-benzoylamino)anilino)-6-methoxy-7-(3-(1-morpholino)propoxy)quinazoline; Fig. 1 A). In in vitro kinase assays using purified recombinant proteins, ZM447439 inhibited Aurora A and B with IC50 values of 110 and 130 nM, respectively (Fig. 1 B). In contrast, the majority of other protein kinases assayed were not inhibited by ZM447439. Based on data from model systems, we predicted that an Aurora inhibitor should prevent cell division and inhibit phosphorylation of histone H3 on serine 10. To determine whether ZM447439 inhibits cell division, a panel of human cell lines was treated with 2 μM ZM447439 for up to 96 h (Fig. 1 C). After 18 h, the vast majority of cells in all the lines had 4N DNA contents. All the lines analyzed then endoreduplicated, accumulating cells with DNA contents greater than 4N, demonstrating that ZM447439 completely inhibits cell division. To determine whether ZM447439 inhibits phosphorylation of histone H3 on serine 10, untreated and ZM447439-treated cells were stained with an anti-phosphohistone H3 antibody (Hsu et al., 2000). In untreated cells, chromosomes stained positive for phosphohistone H3 (Fig. 1 D). However, after brief exposures to ZM447439, phosphohistone H3 was not detectable, demonstrating that ZM447439 does inhibit mitotic phosphorylation of histone H3. Consistent with previous observations (Adams et al., 2001c), the lack of histone H3 phosphorylation did not appear to affect chromosome condensation.

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