Bub1 and aurora B cooperate to maintain BubR1-mediated inhibition of APC/CCdc20

Abstract

The spindle checkpoint maintains genome stability by inhibiting Cdc20-mediated activation of the anaphase promoting complex/cyclosome (APC/C) until all the chromosomes correctly align on the microtubule spindle apparatus via their kinetochores. BubR1, an essential component of this checkpoint, localises to kinetochores and its kinase activity is regulated by the kinesin-related motor protein Cenp-E. BubR1 also inhibits APC/CCdc20 in vitro, thus providing a molecular link between kinetochore-microtubule interactions and the proteolytic machinery that regulates mitotic progression. Several other protein kinases, including Bub1 and members of the Ipl1/aurora family, also regulate anaphase onset. However, in human somatic cells Bub1 and aurora B kinase activity do not appear to be essential for spindle checkpoint function. Specifically, when Bub1 is inhibited by RNA interference, or aurora kinase activity is inhibited with the small molecule ZM447439, cells arrest transiently in mitosis following exposure to spindle toxins that prevent microtubule polymerisation. Here, we show that mitotic arrest of Bub1-deficient cells is dependent on aurora kinase activity, and vice versa. We suggest therefore that the checkpoint is composed of two arms, one dependent on Bub1, the other on aurora B. Analysis of BubR1 complexes suggests that both of these arms converge on the mitotic checkpoint complex (MCC), which includes BubR1, Bub3, Mad2 and Cdc20. Although it is known that MCC components can bind and inhibit the APC/C, we show here for the first time that the binding of the MCC to the APC/C is dependent on an active checkpoint signal. Furthermore, we show that both Bub1 and aurora kinase activity are required to promote binding of the MCC to the APC/C. These observations provide a simple explanation of why BubR1 and Mad2 are essential for checkpoint function following spindle destruction, yet Bub1 and aurora B kinase activity are not. Taken together with other observations, we suggest that these two arms respond to different spindle cues: whereas the Bub1 arm monitors kinetochore-microtubule attachment, the aurora B arm monitors biorientation. This bifurcation in the signalling mechanism may help explain why many tumour cells mount a robust checkpoint response following spindle damage, despite exhibiting chromosome instability.

VX-680 Inhibits Aurora A and Aurora B Kinase Activity in Human Cells

“…DLD-1 and HeLa cells were cultured (as described in ref. 22). HeLa cells were synchronised using a double thymidine block and release protocol.…”

“…DLD-1 cells stably expressing GFP-Histone H2B were exploited to analyse mitosis in living cells by fluorescence and phase-contrast microscopy. 22 Twelve hours after plating in 30 mm glass-bottomed Petri dishes (MatTek), 200 nM VX-680 or DMSO were added to the cell culture medium and DNA morphology was captured by automatic point revisiting of eight independent fields of view. Images were acquired using a Roper 512D EM-CCD camera Cell Cycle 2007; Vol.…”

Histone deacetylase inhibitors and genomic instability

“…A recent study reported that SBHA treatment results in failure to properly recruit the chromosomal passenger complex (CPC) at centromeres. Given that survivin and Aurora B, two essential CPC componants, control SAC protein localization [101][102][103], it was proposed that the HDACI-induced overriding of SAC results from CPC mistargeting [91]. Consistent with a role for HDACI in inhibiting the SAC, a synergistic effect of a combination of microtubule-targeting drugs with TSA has been reported [96].…”

Length of mitotic arrest induced by microtubule-stabilizing drugs determines cell death after mitotic exit

“…1). ZM447439 more efficiently abrogates the mitotic arrest induced by drugs that stabilize microtubules than those that depolymerize microtubules such as nocodazole (20,21). This effect is likely due to a requirement for Aurora kinase-independent pathways in the attachmentsensing arm of the SAC (triggered by loss of microtubules) versus the tension-sensing arm, which is highly dependent on Aurora kinases (3).…”