Caspase-Mediated Specific Cleavage of BubR1 Is a Determinant of Mitotic Progression

Abstract

The fidelity of chromosomal duplication is monitored by cell cycle checkpoints operational during mitosis. One such cell cycle delay is invoked by microtubule-targeting agents such as nocodazole or paclitaxel (Taxol) and is mediated by mitotic checkpoint proteins that include BubR1. Relatively little is known about the regulation of expression and stability of BubR1 (or other checkpoint proteins) and how these factors dictate the durability of the cell cycle delay. We report here that treatment of HeLa cells with spindle-disrupting agents resulted in caspase activation and precipitated the cleavage of BubR1. This mechanism ultimately leads to reduced levels of full-length protein, which are accompanied by abrogation of the mitotic block; the checkpoint abrogation is substantially accelerated by inhibition of de novo protein synthesis. In contrast, inhibition of caspase activity blocked BubR1 degradation and prolonged mitosis. To confirm a direct link between caspase activity and BubR1 protein expression, we identified by site-directed mutagenesis the specific caspase cleavage sites cleaved after exposure to paclitaxel. Surprisingly, BubR1 has two sites of cleavage: primarily at Asp607/Asp610 and secondarily at Asp576/Asp579. BubR1 mutated at both locations (BubR1Δ579Δ610) was resistant to paclitaxel-induced degradation. Expression of BubR1Δ579Δ610 augmented the mitotic delay induced by spindle disruption in transfected cells as well as in clones engineered to inducibly express the mutant protein upon exposure to doxycycline and ultimately led to increased aneuploidy. Underscoring the importance of these caspase cleavage sites, both tetrapeptide motifs are identified in the amino acid sequences of human, mouse, chicken, and Xenopus BubR1. These results are potentially the first to link the control of the stability of a key mitotic checkpoint protein to caspase activation, a regulatory pathway that may be involved in killing defective cells and that has been evolutionarily conserved.

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Caspase-Mediated Specific Cleavage of BubR1 Is a Determinant of Mitotic Progression

ACKNOWLEDGMENTS

We are grateful to other members of the Kao Laboratory for expert assistance, especially K. Ranh Voong and Jessica Liao. We thank Tim J. Yen and Gordon Chan for the gifts of anti-BubR1 and anti-Bub1 antibody and of BubR1 cDNA. We acknowledge the support of W. Gillies McKenna, Anna Kennedy, and Larry Solin. We are grateful to Raimundo Freire for helpful comments and generous gifts of reagents.

S.E.P. was supported by National Institutes of Health Training Grant 5T32CA009677. This work was also supported by the Office of Research and Development Medical Research Service, Department of Veterans Affairs (Advanced Career Research Award), the National Institutes of Health (CA107956-01), and funds from the University of Pennsylvania Research Foundation and the Breast Cancer Research Foundation.